Replikin-based compounds for prevention and treatment of influenza and methods of differentiating infectivity and lethality in influenza

ABSTRACT

The present invention provides methods of differentiating the infectivity and lethality of isolates of influenza virus and provides compounds for diagnosing, preventing, and treating outbreaks of influenza virus including compounds for diagnosing, preventing, and treating across different strains of influenza virus.

This application claims priority to U.S. Provisional Appln. Ser. No.61/246,006, filed Sep. 25, 2009, U.S. application Ser. No. 12/538,027,filed Aug. 7, 2009, U.S. Provisional Appln. Ser. No. 61/185,160, filedJun. 8, 2009, U.S. Provisional Appln. Ser. No. 61/179,686, filed May 19,2009, U.S. Provisional Appln. Ser. No. 61/172,115, filed Apr. 23, 2009,U.S. application Ser. No. 12/429,044, filed Apr. 23, 2009, andPCT/US09/41565, filed Apr. 23, 2009, each of which is incorporatedherein by reference in its entirety. This application furtherincorporates by reference in their entireties, U.S. Provisional Appln.Ser. No. 61/143,618, filed Jan. 9, 2009, U.S. Provisional Appln. Ser.No. 61/087,354, filed Aug. 8, 2008, U.S. Provisional Appln. Ser. No.61/054,010, filed May 16, 2008, U.S. application Ser. No. 12/108,458,filed Apr. 23, 2008, PCT/US2008/61336, filed Apr. 23, 2008, U.S.application Ser. No. 12/010,027, filed Jan. 18, 2008, U.S. ProvisionalAppln. Ser. No. 60/991,676, filed Nov. 30, 2007, U.S. application Ser.No. 11/923,559, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No.60/982,336, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No.60/982,333, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No.60/982,338, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No.60/935,816, filed Aug. 31, 2007, U.S. Provisional Appln. Ser. No.60/935,499 filed Aug. 16, 2007, U.S. Provisional Appln. Ser. No.60/954,743, filed Aug. 8, 2007, U.S. application Ser. No. 11/755,597,filed May 30, 2007, U.S. Provisional Appln. Ser. No. 60/898,097, filedJan. 30, 2007, U.S. Provisional Appln. Ser. No. 60/880,966, filed Jan.18, 2007, U.S. Provisional Appln. Ser. No. 60/853,744, filed Oct. 24,2006, U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006, U.S.application Ser. No. 11/116,203, filed Apr. 28, 2005, U.S. applicationSer. No. 10/860,050, filed Jun. 4, 2004, now U.S. Pat. No. 7,442,761,U.S. application Ser. No. 10/189,437, filed Jul. 8, 2002, now U.S. Pat.No. 7,452,963, U.S. application Ser. No. 10/105,232, filed Mar. 26,2002, now U.S. Pat. No. 7,189,800, U.S. application Ser. No. 09/984,057,filed Oct. 26, 2001, now U.S. Pat. No. 7,420,028, and U.S. applicationSer. No. 09/984,056, filed Oct. 26, 2001, now U.S. Pat. No. 7,176,275,each in its entirety.

FIELD OF THE INVENTION

The present invention relates to therapies for preventing and treatinginfluenza virus, methods of predicting and differentiating infectivityand lethality of influenza outbreaks, and compounds for diagnostic,therapeutic, and/or preventive purposes in influenza.

BACKGROUND OF THE INVENTION

Influenza is an acute respiratory illness of global importance in humansand animals (both domesticated and wild) including, but not limited to,horses, pigs, chickens, ducks, turkeys, ferrets, and wild birds.Virulent and lethal outbreaks of influenza continue to threaten globalhealth. As demonstrated by the H1N1 influenza pandemic of 2009,researchers, government officials, and medical practitioners are acutelyaware of the continuing threat of pandemics of virulent and lethalinfluenza requiring new methods of treatment and novel therapeuticcompounds. Researchers, government officials, and medical practitionershave also believed, however, it was not possible to develop long termtherapies against influenza viruses across strains and across timebecause the influenza virus is subject to such rapid mutation as itmoves through a population (and subject to hosting in a large variety ofnon-human reservoirs), such that an effective therapy for one year in aparticular strain is not expected to be effective in the years to comeagainst that strain or against other strains of influenza virus.Researchers, government officials, and medical practitioners havenevertheless long understood that a therapy against influenza that couldbe applied across strains and/or across time would be immensely helpfulin attacking the global threat of influenza. Such a therapy was simplynot considered possible until now.

As such, until now, influenza vaccines have remained the most effectivedefense against influenza virus. However, because of the ability of thevirus to mutate, and the availability of non-human host reservoirs,influenza has continued to remain an emergent or re-emergent infectiousthreat.

Traditionally, vaccines have been developed on a twice-yearly basis,based on post hoc hematological classification of the increasing numberof emerging influenza virus strains. As such, the only basis for annualclassification of influenza virus as present or absent in a given yearwas identification by serological testing of the hemagglutinin andneuraminidase proteins in an isolate of virus. The activity of a strainof influenza was, as a result, only recorded after the occurrence of anoutbreak, never in advance.

Because of the delay inherent in traditional methods of surveillance,presently applied technology does not allow for the design of effectivevaccines early in an outbreak and has not allowed for the design ofvaccines that might apply to more than one outbreak over time or acrossstrains. Furthermore, presently applied vaccine production technologydelays the availability of vaccines even after an outbreak occurs sincemany months are needed for production of vaccines following vaccinedesign. As previous and current events make clear (such as the currentH1N1 influenza pandemic of 2009), despite the best intentions of thevaccine industry, current biological technology cannot supply all of theworld's 6 billion people and billions of animals in a timely manner withvaccines against emerging diseases. That is, using currently appliedtechnology, vaccines against emerging diseases are not produced prior toglobal outbreak of the disease and often are not produced until theemerging disease outbreak has subsided.

The applicants' discovery of Replikin chemistry in the virus genomestructure, however, now provides methods of predicting future outbreaksof strains of influenza virus and now provides methods of identifyingconserved targets in emerging strains of influenza against whichvaccines may be developed prior to or at the outset of an outbreak. Suchvaccine development can be undertaken in as few as seven days.

When an outbreak of influenza is identified, one aspect of the outbreakthat is useful to public health researchers and government officials isa differentiation of the infectivity and the lethality of the influenzavirus strain that is the agent of the outbreak. An influenza virusstrain that is both relatively more infective and relatively more lethalis an influenza strain that will likely cause increased morbidity andmortality in an outbreak. When public health researchers and governmentofficials have advanced knowledge of the infectivity and lethality of aninfluenza strain, they have crucial additional time for preparations ofvaccines and other health measures in advance of a spreading outbreak.Early differentiation of infectivity and lethality of a strain ofinfluenza that is causing an outbreak is of significant importance andutility to those coordinating a response to the outbreak and to thosedesigning vaccines and other health measures in response to an outbreak.For example, early differentiation of infectivity and lethality of astrain of influenza virus causing an outbreak allows for a design oftherapies that target the infectivity of a virus, the lethality of avirus, or both,

There is a continuing need in the art for quantitative methods ofdifferentiating, preventing, and treating outbreaks caused by virulentstrains of influenza. Because of the annual administration of influenzavaccines and the short period of time when a vaccine can beadministered, strategies directed at improving vaccine coverage are ofcritical importance. There is additionally a continuing need in the artfor therapies against influenza virus that apply across strains andacross time.

Replikin peptides are a family of small peptides that have beencorrelated with the phenomenon of rapid replication in influenza,malaria, West Nile virus, foot and mouth disease, and many otherpathogens. Replikin peptides have likewise been generally correlatedwith the phenomenon of rapid replication in viruses, organisms, andmalignancies.

Identification of Replikin peptides has provided targets for detectionand treatment of pathogens, including vaccine development againstvirulent pathogens such as influenza virus, malaria, West Nile virus,and foot and mouth disease virus. In general, knowledge of andidentification of this family of peptides enables development ofeffective therapies and vaccines for any pathogen that harborsReplikins. The phenomenon of the association of Replikins with rapidreplication and virulence has been fully described in U.S. Pat. No.7,189,800; U.S. Pat. No. 7,176,275; U.S. Pat. No. 7,442,761; and U.S.application Ser. No. 11/355,120. Both Replikin concentration (number ofReplikins per 100 amino acids) and Replikin composition have beencorrelated with the functional phenomenon of rapid replication.

There is a continuing need for monitoring Replikin sequences in strainsof influenza virus to identify compounds for therapies that respond toinfluenza mutations. There is also a need to develop Replikin-basedtherapies that are effective across strains and within strains as theymutate over time. There is an additional need to develop Replikin-basedtherapies that are active against the infectivity of influenza virusesand/or that are active against the lethality of influenza viruses.

SUMMARY OF THE INVENTION

The present invention provides methods of differentiating theinfectivity of an influenza virus isolate or strain of influenza virusfrom the lethality of the influenza virus isolate or strain of influenzavirus and compounds for diagnostic, therapeutic, and/or preventivepurposes in influenza including any strain of influenza.

A first non-limiting aspect of the present invention provides anisolated or synthesized protein fragment, polypeptide, or peptidecomprising at least one peptide A where peptide A is at least 30%, 40%,50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one ofSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In a non-limitingembodiment of the first aspect, the amino acid sequence of the proteinfragment, polypeptide, or peptide partially matches the amino acidsequence of an expressed whole protein wherein at least one, five, ten,twenty, thirty, forty, fifty, one hundred, two hundred, three hundred,four hundred, five hundred or more amino acid residues of the amino acidsequence of the expressed whole protein are not present in the proteinfragment, polypeptide, or peptide. In another non-limiting embodiment ofthe first aspect, the amino acid sequence of said protein fragment,polypeptide, or peptide partially matches the amino acid sequence of anexpressed whole protein wherein at least one, ten, twenty, thirty,forty, fifty, sixty, seventy, eighty, ninety, one hundred, one hundredfifty, two hundred, two hundred fifty, three hundred, three hundredfifty, four hundred, four hundred fifty, five hundred, five hundredfifty or more amino acid residues of the amino acid sequence of at leastone terminus of the expressed whole protein are not present at at leastone terminus of said protein fragment, polypeptide, or peptide.

In a further non-limiting embodiment of the first aspect of the presentinvention, the isolated or synthesized protein fragment, polypeptide, orpeptide consists of 7 to about 50 amino acids comprising at least onepeptide A, wherein said peptide A is at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%, homologous with at least one of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66. In another non-limiting embodiment, theisolated or synthesized protein fragment, polypeptide, or peptideconsists of a peptide A that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 100% homologous with at least one of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66, where the length of peptide A is no more than one,five, ten, twenty, thirty, forty, or fifty amino acid residues longerthan the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 withwhich it is homologous. In a further non-limiting embodiment, peptide Ais no more than one, two, three, four, five, six, seven, eight, nine, orten amino acid residues longer than the sequence of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 with which it is homologous.

In a further non-limiting embodiment of the first aspect of the presentinvention, the isolated or synthesized protein fragment, polypeptide, orpeptide consists of any one of the peptides of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66.

A further non-limiting embodiment provides a peptide consisting of SEQID NO(s): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. A furthernon-limiting embodiment provides a peptide consisting of SEQ ID NO(s):21, 22, 23, 24, 25, 26, 27, or 28.

A further non-limiting embodiment of the first aspect of the inventionprovides an isolated or synthesized protein fragment, polypeptide, orpeptide comprising at least one peptide A, where peptide A is at least30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with atleast one of SEQ ID NO(s): 32-66. Another non-limiting embodimentprovides a peptide consisting of at least one of SEQ ID NO(s): 32-66. Ina further non-limiting embodiment, any peptide of SEQ ID NO(s): 32-66 isprovided as comprised in an immunogenic composition and/or comprised ina vaccine.

Another non-limiting embodiment of the first aspect of the inventionprovides a biosynthetic composition consisting essentially of a peptideof SEQ ID NO(s): 1-66. A further non-limiting embodiment provides abiosynthetic composition consisting of a peptide of SEQ ID NO(s): 1-66.

Another non-limiting embodiment of the first aspect of the inventionprovides a protein fragment, polypeptide, or peptide consistingessentially of at least one of SEQ ID NO(s): 1-20 or SEQ ID NO(s):21-66.

In a non-limiting embodiment, an isolated protein fragment, polypeptide,or peptide is chemically synthesized by solid phase methods.

A second non-limiting aspect of the present invention provides animmunogenic composition comprising at least one protein fragment,polypeptide, or peptide of any one of the above-listed proteinfragments, polypeptides, or peptides. In a non-limiting embodiment ofthe second aspect of the present invention, the immunogenic compoundcomprises at least one peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66. Another non-limiting embodiment provides an immunogeniccomposition comprising at least one peptide of SEQ ID NO(s): 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, and 12 and SEQ ID NO(s): 21, 22, 23, 24, 25, 26,27, and 28.

A third non-limiting aspect of the present invention provides a vaccinecomprising at least one protein fragment, polypeptide, or peptide of anyone of the above-listed protein fragments, polypeptides, or peptides. Ina non-limiting embodiment of the third aspect of the present invention,the vaccine comprises at least one peptide of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66.

A fourth non-limiting aspect of the present invention provides acomposition comprising one or more isolated or synthesized peptides thatare 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with atleast one of the peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66. In a non-limiting embodiment, the composition comprises one ormore isolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66. In another non-limiting embodiment, the compositioncomprises two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, or twenty or more isolated or synthesized peptides of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66. In a non-limiting embodiment, thecomposition comprises at least one of the peptides of SEQ ID NO(s):1-12. In another non-limiting embodiment, the composition comprises amixture of peptides, wherein the mixture comprises isolated orsynthesized peptides of SEQ ID NO(s): 1-12. In another non-limitingembodiment, the composition comprises at least one of the peptides ofSEQ ID NO(s): 1-12 and 21-28. In another non-limiting embodiment, thecomposition comprises a mixture of peptides, wherein the mixturecomprises isolated or synthesized peptides of SEQ ID NO(s): 1-12 and21-28. In a non-limiting embodiment, the composition comprises anapproximately equal molar mixture of the isolated or synthesizedpeptides of SEQ ID NO(s): 1-12 or an approximately equal molar mixtureof the isolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.In a further non-limiting embodiment, the composition comprisesapproximately equal weight of the isolated or synthesized peptides ofSEQ ID NO(s): 1-12 or approximately equal weight of the isolated orsynthesized peptides of SEQ ID NO(s): 1-12 and 21-28.

In another non-limiting embodiment, the composition comprises about 10%by weight SEQ ID NO: 1, about 9% by weight SEQ ID NO: 2, about 10% byweight SEQ ID NO: 3, about 6% by weight SEQ ID NO: 4, about 8% by weightSEQ ID NO: 5, about 8% by weight SEQ ID NO: 6, about 7% by weight SEQ IDNO: 7, about 6% by weight SEQ ID NO: 8, about 10% by weight SEQ ID NO:9, about 8% by weight SEQ ID NO: 10, about 7% by weight SEQ ID NO: 11,and about 11% by weight SEQ ID NO: 12.

A fifth aspect of the present invention provides a vaccine comprisingone or more isolated or synthesized peptides that are 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% or more homologous with at least one of thepeptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In anon-limiting embodiment, the vaccine comprises one or more isolated orsynthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. Inanother non-limiting embodiment, the vaccine comprises two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or moreisolated or synthesized peptides of SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66. In a non-limiting embodiment, the vaccine comprises at leastone of the peptides of SEQ ID NO(s): 1-12. In another non-limitingembodiment, the vaccine comprises a mixture of peptides, wherein themixture comprises isolated or synthesized peptides of SEQ ID NO(s):1-12. In another non-limiting embodiment, the vaccine comprises at leastone of the peptides of SEQ ID NO(s): 1-12 and 21-28. In anothernon-limiting embodiment, the vaccine comprises a mixture of peptides,wherein the mixture comprises isolated or synthesized peptides of SEQ IDNO(s): 1-12 and 21-28. In a non-limiting embodiment, the vaccinecomprises an approximately equal molar mixture of the isolated orsynthesized peptides of SEQ ID NO(s): 1-12 or an approximately equalmolar mixture of the isolated or synthesized peptides of SEQ ID NO(s):1-12 and 21-28. In a further non-limiting embodiment, the vaccinecomprises approximately equal weight of the isolated or synthesizedpeptides of SEQ ID NO(s): 1-12 or approximately equal weight of theisolated or synthesized peptides of SEQ ID NO(s): 1-12 and 21-28.

In another non-limiting embodiment, the vaccine comprises about 10% byweight SEQ ID NO: 1, about 9% by weight SEQ ID NO: 2, about 10% byweight SEQ ID NO: 3, about 6% by weight SEQ ID NO: 4, about 8% by weightSEQ ID NO: 5, about 8% by weight SEQ ID NO: 6, about 7% by weight SEQ IDNO: 7, about 6% by weight SEQ ID NO: 8, about 10% by weight SEQ ID NO:9, about 8% by weight SEQ ID NO: 10, about 7% by weight SEQ ID NO: 11,and about 11% by weight SEQ ID NO: 12. In a further non-limitingembodiment, the vaccine comprises a pharmaceutically acceptable carrierand/or adjuvant. In a further non-limiting embodiment, the vaccine isfor the treatment or prevention of influenza virus infection. In afurther non-limiting embodiment, the vaccine is directed against H1N1,H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, orany other strain of influenza A.

A sixth non-limiting aspect of the invention provides an antibody,antibody fragment, or binding agent that binds to at least a portion ofan amino acid sequence of at least one protein fragment, polypeptide, orpeptide comprising a peptide A, wherein the peptide A is 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% or more homologous with at least one of thepeptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In anon-limiting embodiment of the sixth non-limiting aspect, the antibody,antibody fragment, or binding agent binds to at least a portion of anamino acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ormore homologous with at least one of the peptides of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66. In another non-limiting embodiment, theantibody, antibody fragment, or binding agent binds to at least aportion of an amino acid sequence of at least one of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66.

A seventh non-limiting aspect of the present invention provides anisolated or synthesized polypeptide or peptide comprising a peptide Athat has about the same number of amino acid residues as a peptide B,where peptide B is one of the peptides of SEQ ID NO: 1-28, and where thelysine residues and histidine residues in peptide A are conserved ascompared to the lysine residues and histidine residues in peptide B,wherein said isolated or synthesized polypeptide or peptide furthercomprises up to 100 more amino acid residues than does peptide A, andwherein said up to 100 more amino acid residues of said isolated orsynthesized polypeptide or peptide are positioned to the amino-terminusand/or carboxy-terminus of the lysine or histidine termini of peptide A.In a non-limiting embodiment of the seventh aspect of the presentinvention, the up to 100 more amino acid residues is up to one, two,three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty,or fifty more amino acid residues. In a further non-limiting embodiment,the isolated or synthesized polypeptide or peptide consists of peptideA.

A further non-limiting embodiment of the seventh aspect of the presentinvention provides an isolated or synthesized peptide consisting of:

(1) a peptide consisting of about 26 amino acid residues with ahistidine residue within 5 residues of the amino-terminus of the peptidewherein the histidine residue is considered to reside at position 1, andwherein relative to position 1 there is a lysine residue at position 8,a histidine residue at position 10, a lysine residue at position 13, alysine residue at position 18, and a lysine residue at position 26, andwherein up to five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 26;(2) a peptide consisting of about 19 amino acid residues with a lysineresidue within 5 residues of the amino-terminus of the peptide whereinthe lysine residue is considered to reside at position 1, and whereinrelative to position 1 there is a histidine residue at position 3, alysine residue at position 6, a lysine residue at position 11, and alysine residue at position 19, and wherein up to five additionalresidues may be present on the carboxy-terminus of the peptide after thelysine residue at position 19;(3) a peptide consisting of about 29 amino acids residues with a lysineresidue within 5 residues of the amino-terminus of the peptide whereinthe lysine residue is considered to reside at position 1, and whereinrelative to position 1 there is a lysine residue at position 2, a lysineresidue at position 10, a histidine residue at position 28, and ahistidine residue at position 29, and wherein up to five additionalresidues may be present on the carboxy-terminus of the peptide after thehistidine residue at position 29;(4) a peptide consisting of about 27 amino acid residues with ahistidine residue within 5 residues of the amino-terminus of the peptidewherein the histidine residue is considered to reside at position 1, andwherein relative to position 1 there is a histidine residue at position2, a lysine residue at position 14, a lysine residue at position 19, anda lysine residue at position 27, and wherein up to five additionalresidues may be present on the carboxy-terminus of the peptide after thelysine residue at position 27.(5) a peptide consisting of about 21 amino acid residues with ahistidine residue within 5 residues of the amino-terminus of the peptidewherein the histidine residue is considered to reside at position 1 andwherein relative to position 1 there is a lysine residue at position 6,a lysine residue at position 11, and a lysine residue at position 21,and wherein up to five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 21;(6) a peptide consisting of about 22 amino acid residues with a lysineresidue within 5 residues of the amino-terminus of the peptide whereinthe lysine residue is considered to reside at position 1, and whereinrelative to position 1 there is a lysine residue at position 11, and ahistidine residue at position 22, and wherein up to five additionalresidues may be present on the carboxy-terminus of the peptide after thehistidine residue at position 22;(7) a peptide consisting of about 17 amino acids with a lysine residuewithin 5 residues of the amino-terminus of the peptide wherein thelysine residue is considered to reside at position 1, and whereinrelative to position 1 there is a lysine residue at position 9, and ahistidine residue at position 17, and wherein up to five additionalresidues may be present on the carboxy-terminus of the peptide after thehistidine residue at position 17;(8) a peptide consisting of about 15 amino acid residues with ahistidine residue within 5 residues of the amino-terminus of the peptidewherein the histidine residue is considered to reside at position 1, andwherein relative to position 1 there is a lysine residue at position 5,a lysine residue at position 14, and a lysine residue at position 15,and wherein up to five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 15;(9) a peptide of about 18 amino acid residues with a lysine residuewithin 5 residues of the amino-terminus of the peptide wherein thelysine residue is considered to reside at position 1, and whereinrelative to position 1 there is a lysine residue at position 2, ahistidine residue at position 5, a lysine residue at position 6, alysine residue at positions 11, 12, and 13, and a lysine residue atposition 18, and wherein up to five additional residues may be presenton the carboxy-terminus of the peptide after the lysine residue atposition 18;(10) a peptide of about 14 amino acid residues with a histidine residuewithin 5 residues of the amino-terminus of the peptide wherein thehistidine residue is considered to reside at position 1, and whereinrelative to position 1 there is a lysine residue at position 2, a lysineresidue at positions 7, 8, and 9, and a lysine residue at position 14,and wherein up to five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 14;(11) a peptide of about 26 amino acid residues with a histidine residuewithin 5 residues of the amino-terminus of the peptide wherein thehistidine residue is considered to reside at position 1, and whereinrelative to position 1 there is a lysine residue at position 16, and alysine residue at positions 24, 25, and 26, and wherein up to fiveadditional residues may be present on the carboxy-terminus of thepeptide after the lysine residue at position 26; or(12) a peptide consisting of about 35 amino acid residues with ahistidine residue within 5 residues of the amino terminus of the peptidewherein the histidine residue is considered to reside at position 1, andwherein relative to position 1 there is a lysine residue at position 6,a lysine residue at position 28, and a lysine residue at position 35,and wherein up to five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 35.

In a further non-limiting embodiment, the isolated or synthesizedpeptide has an amino-terminus at position 1 and has a carboxy-terminusat the amino acid residue for which a position is expressly numberedthat is the farthest to the carboxy-terminus of the peptide.

An eighth non-limiting aspect of the present invention provides a methodof making a vaccine comprising: selecting at least one isolated orsynthesized protein fragment, polypeptide, or peptide comprising atleast one peptide A, where peptide A is at least 30%, 40%, 50%, 60%,70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66 as a component of a vaccine; andmaking said vaccine. In a non-limiting embodiment, the method of makinga vaccine comprises: selecting at least one isolated or synthesizedpeptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 as at least onecomponent; and making said vaccine with the at least one component.

In another non-limiting embodiment, the method of making a vaccinecomprises selecting at least two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, or more isolated or synthesized peptides ofSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 as the at least onecomponent of said vaccine. In another non-limiting embodiment, the atleast one isolated or synthesized protein fragment, polypeptide, orpeptide has the same amino acid sequence as at least one proteinfragment, polypeptide or peptide identified in an emerging strain ofinfluenza virus up to six months, one year, two years, or three yearsprior to making said vaccine.

A ninth non-limiting aspect of the present invention provides a methodfor preventing or treating influenza virus infection comprisingadministering at least one isolated or synthesized protein fragment,polypeptide, or peptide comprising at least one peptide A, where peptideA is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%,homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 to an animal or human. In a non-limiting embodiment, the at leastone isolated or synthesized protein fragment, polypeptide, or peptideconsists of at least one peptide A at least 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95% or more homologous with at least one of the peptidesSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In another non-limitingembodiment, the at least one isolated or synthesized peptide of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66 is administered to an animal orhuman. In another non-limiting embodiment of the invention, at least oneagent is capable of binding at least a portion of said peptide A that isat least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologouswith at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

A tenth non-limiting aspect of the present invention provides anisolated or synthesized nucleic acid sequence that encodes a proteinfragment, polypeptide, or peptide comprising at least one peptide A,where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66. In a non-limiting embodiment, the isolated or synthesizednucleic acid sequence encodes for a peptide consisting of 7 to about 50amino acid residues and comprising any one or more of the peptidesequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In anothernon-limiting embodiment, the nucleic acid sequence encodes for a peptidethat is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or morehomologous with at least one of the peptide sequences of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66. In another non-limiting embodiment, thenucleic acid sequence encodes for a peptide that consists of at leastone of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66.

In another non-limiting embodiment of the tenth aspect of the presentinvention, the isolated or synthesized nucleic acid sequence iscomprised in an immunogenic compound. In another non-limitingembodiment, the isolated or synthesized nucleic acid sequence iscomprised in a vaccine.

Another non-limiting embodiment of the tenth aspect of the presentinvention provides an isolated or synthesized nucleic acid sequence thatis antisense to a nucleic acid that encodes for a peptide that is atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous withat least one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66. Another non-limiting embodiment provides a smallinterfering nucleic acid sequence that is about 10 to about 50 nucleicacids in length and is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or morehomologous with a nucleic acid that encodes for any portion of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66 or is 30%, 40%, 50%, 60%, 70%, 80%,90% or more homologous with a nucleic acid that is antisense to anucleic acid that encodes for any portion of one of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66. In another non-limiting embodiment the smallinterfering nucleic acid sequences is about 15 to about 45, about 20 toabout 30, or about 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleic acidsin length.

An eleventh non-limiting aspect of the present invention, provides for avaccine comprising at least one protein fragment, polypeptide, orpeptide comprising at least one peptide A, where peptide A is at least30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with atleast one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from ahemagglutinin protein area of influenza virus, or a synthesized versionthereof, and at least one protein fragment, polypeptide, or peptidecomprising at least one peptide A, where peptide A is at least 30%, 40%,50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one ofSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from a protein orpeptide encoded by a pB1 gene area of influenza virus, or a synthesizedversion thereof. In a non-limiting embodiment, the at least one proteinfragment, polypeptide, or peptide is isolated from an isolate ofinfluenza virus predicted to have a greater infectivity than at leastone other isolate of influenza virus and the at least one proteinfragment, polypeptide, or peptide isolated from the pB1 gene area, orsynthesized version thereof, is isolated from an isolate of influenzavirus predicted to have a greater lethality than at least one otherisolate of influenza virus. In another non-limiting embodiment, the atleast one protein fragment, polypeptide, or peptide isolated from thehemagglutinin protein area, or synthesized version thereof, is aplurality of protein fragments, polypeptides, and/or peptides isolatedfrom the hemagglutinin protein area and the at least one proteinfragment, polypeptide, or peptide isolated from the pB1 gene area, orsynthesized version thereof, is a plurality of protein fragments,polypeptides, and/or peptides isolated from the pB1 gene area.

In a non-limiting embodiment, the at least one protein fragment,polypeptide, or peptide isolated from the hemagglutinin protein area, orsynthesized version thereof, is at least one Replikin peptide isolatedfrom the hemagglutinin protein area and the at least one proteinfragment, polypeptide, or peptide isolated from the pB1 gene area, orsynthesized version thereof, is at least one Replikin peptide isolatedfrom the pB1 gene area. In a non-limiting embodiment, the at least oneReplikin peptide isolated from a hemagglutinin protein area, orsynthesized version thereof, is a plurality of Replikin peptidesisolated from a hemagglutinin protein area and the at least one Replikinpeptide isolated from a pB1 gene area, or synthesized version thereof,is a plurality of Replikin peptides isolated from a pB1 gene area. In anon-limiting embodiment, the plurality of Replikin peptides isolatedfrom a hemagglutinin protein area, or synthesized version thereof, is aplurality of the shortest Replikin peptides identified in an influenzavirus isolate or a plurality of influenza virus isolates predicted tohave a greater infectivity than at least one other isolate of influenzavirus and said plurality of Replikin peptides isolated from a pB1 genearea, or synthesized version thereof, is a plurality of the shortestReplikin peptides identified in an influenza virus isolate or aplurality of influenza virus isolates predicted to have a greaterlethality than at least one other isolate of influenza virus.

In a non-limiting embodiment, the vaccine is directed against influenzaA, influenza B, or influenza C. In a further non-limiting embodiment,the vaccine is directed against H1N1, H1N2, H2N2, H3N2, H3N8, H5N1,H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain of influenza Avirus.

A twelfth non-limiting aspect of the present invention provides a methodof making a vaccine comprising: selecting at least one peptide A,wherein said peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66 isolated from a hemagglutinin protein area (or asynthesized version thereof) as a component of said vaccine; andselecting at least one peptide B, wherein said peptide B is at least30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with atleast one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from apB1 gene area (or a synthesized version thereof) as a component of saidvaccine and making said vaccine comprising said components.

In a non-limiting embodiment, a method of making a vaccine comprises:identifying (1) at least one protein, protein fragment, polypeptide, orpeptide of a hemagglutinin protein area in or derived from an isolate ofinfluenza virus having relatively greater infectivity than anotherisolate of influenza virus or a plurality of isolates of influenzaviruses, and (2) at least one protein, protein fragment, polypeptide, orpeptide of a pB1 gene area in or derived from an isolate of influenzavirus having relatively greater lethality than another isolate ofinfluenza virus or a plurality of isolates of influenza virus; andcombining said at least one protein, protein fragment, polypeptide, orpeptide of a hemagglutinin protein area and said at least one protein,protein fragment, polypeptide, or peptide of a pB1 gene area to form avaccine.

In another non-limiting embodiment of the twelfth aspect of the presentinvention, a method of differentiating the relative infectivity ofisolate A of influenza virus or a plurality of isolates A of influenzavirus from the relative infectivity of isolate B of influenza virus or aplurality of isolates B of influenza virus and the relative lethality ofisolate A of influenza virus or a plurality of isolates A of influenzavirus from the relative lethality of isolate B of influenza virus or aplurality of isolates B of influenza virus is provided comprising:

comparing the Replikin Count of the hemagglutinin protein area ofisolate A or the mean Replikin Count of the hemagglutinin protein areasof a plurality of isolates A to the Replikin Count of the hemagglutininprotein area of isolate B or the mean Replikin Count of thehemagglutinin protein area of a plurality of isolates B;

comparing the Replikin Count of the pB1 gene area of isolate A or themean Replikin Count of the pB1 gene area of a plurality of isolates A tothe Replikin Count of the pB1 gene area of isolate B or the meanReplikin Count of the pB1 gene area of a plurality of isolates B; and

differentiating the relative infectivity of isolate A or a plurality ofisolates A from the relative infectivity of isolate B or a plurality ofisolates B and the relative lethality of isolate A or a plurality ofisolates A from the relative lethality of isolate B or a plurality ofisolates B.

In another non-limiting embodiment, the isolate A or the plurality ofisolates A is from a different region or time from the isolate B or theplurality of isolates B.

Another non-limiting embodiment provides a method of differentiating apredicted future relative infectivity of at least one strain A ofinfluenza virus as compared to a time T₀ from a predicted futurerelative lethality of said at least one strain A of influenza virus ascompared to time T₀ comprising:

comparing a trend of Replikin Counts in the hemagglutinin protein areaof a plurality of isolates of strain A ending at time T₀, wherein saidisolates are isolated at different time periods including time T₀, to atrend of Replikin Counts in the pB1 gene area of a plurality of isolatesof strain A ending at time T₀, wherein said isolates are isolated atdifferent time periods including time T₀, and

differentiating the future relative infectivity of said at least onestrain A from the future relative lethality of said at least one strainA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an immune response with protective effect followingadministration of a vaccine comprising a mixture of peptides of SEQ IDNO(s): 1-12 to chickens later challenged with Low-Path H5N1 virus.Eighty chickens were divided into four groups of twenty chickens each ona first day after hatch. Group 1 was a negative control subjected toneither vaccination nor infection with the Low-Path H5N1 virus. Group 2was a vaccine control subjected to vaccination intranasally on day 1after hatch, intraocularly on day 7 after hatch, and via sprayinhalation on day 14 after hatch. Group 2 was not subject to infectionwith the Low-Path H5N1 virus. Group 3 was subjected to vaccination onthe same schedule as Group 2 and Low-Path H5N1 was introduced to thesoft palate of the chickens on day 28. Group 4 was a challenged controlthat was not vaccinated but was infected with H5N1 on day 28 via thesoft palate. On the seventh, fourteenth, and twenty-first days followingchallenge on day 28, between six and nine chickens from each group weretested for serum production of antibodies against H5N1 virus. The datafrom the serum antibody tests are contained in Table 1 and illustratedin FIG. 1. FIG. 1 illustrates that only one of seven (14%) chickenstested in Group 3 (vaccinated and challenged with virus) was observed toproduce antibody in serum seven days after challenge while four of sevenchickens (57%) tested in Group 4 (not vaccinated but challenged) wasobserved to produce antibody in serum seven days after challenge. FIG. 1further illustrates that only three of six chickens (50%) tested inGroup 3 were observed to produce antibody in serum fourteen days afterchallenge while seven of nine (78%) chickens tested in Group 4 wereobserved to produce antibody in serum fourteen days after challenge.FIG. 1 further illustrates that two of seven (29%) chickens tested inGroup 3 were observed to produce antibody in serum twenty-one days afterchallenge while three of nine (33%) of chickens tested in Group 4 wereobserved to produce antibody in serum twenty-one days after challenge.In the vaccine control (Group 2), six of six tested chickens (100%) wereobserved to produce antibody in serum 14 days after challenge while nochickens tested on day 7 or 21 following challenge were observed toproduce antibody in serum. In the negative control (Group 1), nochickens were observed to produce antibody in serum on any day oftesting. In combination with data provided in Table 2 (in Example 1below), which demonstrates that no H5N1 virus was observed by PCRdetection in feces or saliva for chickens in Groups 1, 2, and 3(negative control, vaccine control, a vaccine/challenge groups,respectively) and that H5N1 virus was observed by PCR detection in fecesand saliva for all chickens in Group 4 (challenge control), one ofordinary skill in the art concludes that chickens in the vaccinated andchallenged group (Group 3) were provided a measure of protection fromthe challenge with Low-Path H5N1 on day 28 following hatch.

FIG. 2 illustrates a double differentiation between the infectivity andthe lethality of isolates of H5N1 isolated between 2004 and 2008. InFIG. 2, the black columns represent the mean annual Replikin Count forhemagglutinin protein area sequences of isolates of H5N1 influenza viruspublicly available at www.pubmed.com for a given year between 2004 and2008. Standard deviation is denoted by the capped line on top of theblack columns. The hemagglutinin protein area is associated withinfectivity in influenza. The gray columns represent the mean annualReplikin Count for sequences from the pB1 gene area of isolates of H5N1influenza virus publicly available at www.pubmed.com for a given yearbetween 2004 and 2008. Standard deviation is denoted by the capped lineon top of the gray columns. The pB1 gene area of influenza is associatedwith lethality in influenza. The data for FIG. 2 is disclosed in Table 3in Example 2 below. FIG. 2 illustrates that Replikin Count for thehemagglutinin protein area is differentiable from Replikin Count for thepB1 gene area and that infectivity properties in H5N1 are differentiablefrom lethality properties in H5N1. The data in FIG. 2 corresponds toepidemiological data in H5N1. Human mortality (related to the lethalityproperty of the pB1 gene area) has increased in H5N1 from 1997 throughat least 2007, when mortality rates reached as high as 80% in Indonesia.Mortality rates have remained high since then with the World HealthOrganization estimating a mortality rate of at least 60% in the currentoutbreak of H5N1 influenza. Infectivity rates, on the other hand, haveremained very low in H5N1 with highly limited possible human-to-humantransmission.

FIG. 3 illustrates a double differentiation between the infectivity andthe lethality of isolates of H1N1 isolated between 2004 and May 18,2009. In FIG. 3, the black columns represent the mean annual ReplikinCount for hemagglutinin protein area sequences (associated withinfectivity) publicly available at www.pubmed.com for isolates of H1N1influenza in a given year between 2004 and 2009. Standard deviation isdenoted by the capped line on top of the black columns. The gray columnsrepresent the mean annual Replikin Count for sequences from the pB1 genearea (associated with lethality) publicly available at www.pubmed.comfor isolates of H1N1 influenza in a given year between 2004 and 2009.Standard deviation is denoted by the capped line on top of the graycolumns. The data for FIG. 3 is disclosed in Table 4 in Example 3 below.FIG. 3 illustrates that Replikin Count for the hemagglutinin proteinarea is differentiable from Replikin Count for the pB1 gene area andthat infectivity properties in H1N1 are differentiable from lethalityproperties in H1N1. The data in FIG. 3 corresponds to epidemiologicaldata in H1N1. Infectivity in H1N1 has increased dramatically in 2009resulting in a global outbreak of H1N1 influenza apparently beginning inor near Mexico or the southwestern United States around the spring of2009. The increase in Replikin Count in the hemagglutinin protein areaof isolates of H1N1 in the winter of 2008 allowed for an April 2008prediction of the current global outbreak in 2009. Additionally,lethality in H1N1 has been observed to remain generally low between 2004and 2009 with a spike in lethality in early 2009.

FIG. 4 illustrates a double differentiation between the infectivity andthe lethality of isolates of H1N1 isolated between 2001 and Jun. 8,2009. In FIG. 4, black columns represent the mean annual Replikin Countfor hemagglutinin protein area sequences (associated with infectivity)of isolates of H1N1 influenza publicly available at www.pubmed.com for agiven year between 2001 and 2009 (the 2009 column represents the meanannual Replikin Count for hemagglutinin protein area sequences ofisolates of H1N1 influenza publicly available from Jan. 1, 2009 throughJun. 8, 2009). Standard deviation is denoted by the capped line on topof the black columns. Gray columns represent the mean annual ReplikinCount for sequences from the pB1 gene area (associated with lethality)of isolates of H1N1 influenza publicly available at www.pubmed.com for agiven year between 2001 and 2009 (the 2009 column represents the meanannual Replikin Count for the pB1 gene area sequences of isolates ofH1N1 influenza publicly available from Jan. 1, 2009 through Jun. 8,2009). Standard deviation is denoted by the capped line on top of thegray columns. The data for FIG. 4 is disclosed in Tables 5 and 6 inExample 4 below. FIG. 4 illustrates that Replikin Count for thehemagglutinin protein area is differentiable from Replikin Count for thepB1 gene area and that infectivity properties in H1N1 are differentiablefrom lethality properties in H1N1. The data in FIG. 4 corresponds toepidemiological data in H1N1. As described above, infectivity in H1N1increased dramatically in 2009 resulting in an H1N1 pandemic.Additionally, lethality in H1N1 has been observed to remain generallylow between 2004 and 2008 with a spike in lethality in 2009 based ondata analyzed between May 18 and Jun. 8, 2009. The spike in lethality in2009 is observed as statistically significant with a p-value of lessthan 0.001. See Table 6 in Example 4 below. An earlier rise in theReplikin Count in the pB1 gene area in 2005 is not statisticallysignificant with a p-value of less than 0.40. See Table 6 below.

FIG. 5 illustrates a double differentiation between the infectivity andthe lethality of isolates of H1N1 isolated between 2001 and Sep. 23,2009. In FIG. 5, the white columns represent the mean annual ReplikinCount for hemagglutinin protein area sequences (publicly available atwww.pubmed.com) of isolates of H1N1 influenza isolated in a given yearfor years 2001 through 2007 and represent the mean Replikin Count fromthe beginning of a given year through to the given date for years 2008and 2009. Standard deviation is denoted by a capped line on top of eachwhite column. The hemagglutinin protein area is associated withinfectivity in influenza. The black columns represent the mean annualReplikin Count for sequences from the pB1 gene area (publicly availableat www.pubmed.com) of isolates of H1N1 influenza isolated in a givenyear for years 2001 through 2007 and represent the mean Replikin Countfrom the beginning of a given year through to the given date for years2008 and 2009. Standard deviation is denoted by a capped line on top ofeach black column. The pB1 gene area of influenza is associated withlethality in influenza. The data for FIG. 5 is disclosed in Table 7 inExample 5 below. FIG. 5 illustrates that Replikin Count for thehemagglutinin protein area is differentiable from Replikin Count for thepB1 gene area and that infectivity properties in H1N1 are differentiablefrom lethality properties in H1N1. The data in FIG. 5 corresponds toepidemiological data. As described above, infectivity in H1N1 increaseddramatically in 2009 resulting in a global outbreak of H1N1 influenzaapparently beginning in or near Mexico or the southwestern United Statesaround the spring of 2009. In further correspondence to FIG. 5, thelethality of the 2009 H1N1 outbreak has been fairly low with theproportion of deaths in the United States attributable to pneumonia andinfluenza below the epidemic threshold. See CDC FluView, Week 36 endingSep. 12, 2009 available at http://www.cdc.gov/flu/weekly/. Further, theCDC has reported that pediatric mortality experienced a peak in June2009, which was followed by a sharp drop in H1N1 pediatric mortalitythrough September 2009. See id. All of this data corresponds to theReplikin Count data provided in FIG. 5.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “protein fragment” as used in this specification is any portion of anexpressed whole protein. A protein fragment may reflect an expressedwhole protein with one or more amino acids removed from the amino acidsequence of the expressed whole protein. A protein fragment may alsoreflect an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100% homologous with any portion of an expressed wholeprotein. A “polypeptide,” as used in this specification, is any portionof a protein fragment and is less than an expressed whole protein.

A “whole protein” or an “expressed whole protein” as used in thisspecification reflect a protein that is expressable from an intact geneof an influenza virus from a start codon to a stop codon. A wholeprotein or expressed whole protein may also reflect a whole protein orexpressed whole protein that has been subject to cellular processing tocreate a protein that is capable of functioning within the virusreplication system in a proper manner for virus replication. A proteinfragment, polypeptide, or peptide “partially matches” the amino acidsequence of an expressed whole protein when the protein fragment,polypeptide, or peptide shares substantially homology with the expressedwhole protein but at least one of the amino acids of the expressed wholeprotein are not present in the protein fragment, polypeptide, orpeptide. “Homologous” or “homology” or “sequence identity” as used inthis specification indicate an amino acid sequence or nucleic acidsequence exhibits substantial structural equivalence with anothersequence, namely any one of SEQ ID NO(s): 1-66 (for purposes of thisparagraph, the basis sequences) or any nucleotide sequence encoding SEQID NO(s): 1-66 (a redundancy in a coding sequence may be consideredidentical to a sequence encoding the same amino acid). To determine thepercent identity or percent homology of an identified sequence, thesequence is aligned for optimal comparison purposes with any one of thebasis sequences. Where gaps are necessary to provide optimal alignment,gaps may be introduced in the identified sequence or in the basissequence. When a position in the identified sequence is occupied by thesame amino acid residue or same nucleotide as the corresponding positionin the basis sequence, the molecules are considered identical at thatposition (as used herein amino acid or nucleic acid “identity” isequivalent to amino acid or nucleic acid “homology”). To determinepercent homology, the amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are comparedbetween the identified sequence and the basis sequence. The total numberof amino acid residues or nucleotides in the identified sequence thatare identical with amino acid residues or nucleotides in the basissequence is divided by the total number of residues or nucleotides inthe basis sequence (if the number of residues or nucleotides in thebasis sequence is greater than the total number of residues ornucleotides in the identified sequence) or by the total number of aminoacid residues or nucleotides in the identified sequence (if the numberof residues or nucleotides in the identified sequence is greater thanthe total number of residues or nucleotides in the basis sequence). Thefinal number is determined as a percentage. As such, the percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps (where a gap must be introduced for optimal alignment ofthe two sequences) and the length of each gap. Any structural orfunctional differences between sequences having sequence identity orhomology will not affect the ability of the sequence to function asindicated in the desired application.

For example, SEQ ID NO: 1 (HAQDILEKEHNGKLCSLKGVRPLILK) is consideredmore than 86% homologous with the following sequenceHAQDILEKEHNGKLCSLKGVRPX_(n=4)LILK (SEQ ID NO: 29). The more than 86%homology between SEQ ID NO: 1 and SEQ ID NO: 29 is determined asfollows: SEQ ID NO: 29 is the identified sequence. SEQ ID NO: 1 is thebasis sequence. Upon alignment, SEQ ID NO: 29 is identical to SEQ ID NO:1 in all 26 residues of SEQ ID NO: 1 (with a gap introduced for the fourresidues represented by X_(n-4)). To determine percent homology, then,the 26 aligned identical residues are divided by the total number ofresidues in SEQ ID NO: 29, namely 30 residues, giving 0.867 or more than86% homology.

As another example, SEQ ID NO: 1 is more than 86% homologous withHAQDXILEKEHNGKLCXSLKGVRXXPLILK (SEQ ID NO: 30) because it is identicalto SEQ ID NO: 30 in all residues except for the residues represented bythe four X residues.

In a further example, SEQ ID NO: 2 (KEHNGKLCSLKGVRPLILK) is more than68% homologous with KEHNGKLCSLKGK (SEQ ID NO: 31). SEQ ID NO: 2 is thebasis sequence and has 19 residues. SEQ ID NO: 31 is the referencesequence and has 13 residues that are identical to SEQ ID NO: 2 butVRPLIL is not present between the glycine at position 12 and theterminal lysine at position 13 (all of the other residues areidentical). To determine percent homology, then, the 13 alignedidentical residues are divided by the total number of residues in SEQ IDNO: 2, namely 19 residues, giving 0.684 or more than 68% homology.

To determine homology between an identified sequence that is containedin a larger polypeptide, protein fragment, or protein, and a basissequence, the polypeptide, protein fragment, or protein must first beoptimally aligned with the basis sequence. Upon alignment of thesequences, the residue in the identified sequence that is farthest tothe amino-terminus of the polypeptide, protein fragment, or protein andidentical to a residue in the basis sequence that is farthest to theamino-terminus of the basis sequence is considered the amino-terminalresidue of the identified sequence. Likewise, upon alignment, theresidue in the identified sequence that is farthest to thecarboxy-terminus of the polypeptide, protein fragment, or protein andidentical to a residue in the basis sequence that is farthest to thecarboxy-terminus of the basis sequence is considered thecarboxy-terminal residue of the identified sequence.

An amino acid sequence of a protein fragment, polypeptide, or peptide is“derived from” an identified protein or gene area of an influenza virus(such as a hemagglutinin protein area or a pB1 gene area) if one ofordinary skill in the art would understand from the structure, history,or other relevant information of the amino acid sequence that itoriginated from an amino acid sequence of the identified protein or genearea of influenza. Among other methods, one of ordinary skill may employanalysis of the homology of the amino acid sequence with the identifiedprotein or gene area. One of ordinary skill may also employ the historyof research used in developing the amino acid sequence to determine thatthe amino acid sequence is derived from an original sequence of theidentified protein or gene area. One of ordinary skill would understandthat a protein fragment, polypeptide, or peptide is derived from anidentified protein, polypeptide, or peptide if it is traceable to theidentified protein, polypeptide, or peptide, if it is deducible orinferable from the identified protein, polypeptide, or peptide, if theidentified protein, polypeptide, or peptide is the source of thepeptide, or if the protein fragment, polypeptide, or peptide is derivedfrom the identified protein, polypeptide, or peptide as understood byone of skill in the art. One of ordinary skill may employ any methodknown now or hereafter for determining whether an amino acid sequence isderived from an identified protein or gene area of an influenza virus.

As used herein, “transmission” means, the movement of a pathogen by anymeans from one animal host to any neighboring animal host.

As used herein, “reservoir” means, a collection of animals, one or allof which are infected with a particular infectious agent, wherein thecollection of animals continues to provide a source of infection outsideof the collection of animals. A reservoir is self-perpetuating andpermits time for modification of viruses within the reservoir andpassing of viruses, including modified viruses, to hosts outside of thereservoir.

As used herein, “concomitant” or “concomitantly” or related wordsreflect a difference between the change in infectivity and the change inlethality in a strain of influenza or in different strains or isolatesof influenza within a particular time period or at a particular timepoint or within a particular region. For example, if the relativeinfectivity of a first isolate from a given time period or time point orfrom a particular region is greater than the relative infectivity of asecond isolate from the same time period or same time point or sameparticular region and the relative lethality of the first isolate is notgreater than the relative lethality of the second isolate, then thelethality of the first isolate is not concomitantly greater than therelative lethality of the second isolate. Additionally, for example, anincrease in the relative infectivity over time in a group of isolatesfrom a particular time period or region that is not accompanied by,attended by, or does not correspond with an increase in the relativelethality over time in the same group of isolates is an increase ininfectivity that is not concomitant with an increase in lethality in thesame group of isolates. Changes in infectivity that are not concomitantwith changes in lethality in a strain of influenza virus allow for thedifferentiation of the properties of infectivity and lethality in astrain of influenza over a particular time period or across differentregions.

As used herein a “vaccine” is any substance, compound, composition,mixture, or other therapeutic substance that, when administered to ahuman or animal via any method of administration known to the skilledartisan now or hereafter, produces an immune response, an antibodyresponse, or a protective effect in the human or animal.

A protein area or a gene area of an influenza protein or gene is theprotein or gene of influenza as known to one of skill in the art.Because one skilled artisan may choose to identify a first terminus of aprotein or gene in influenza at a different starting point than anotherskilled artisan and one skilled artisan may choose to identify a secondterminus of a protein or gene in influenza at a different ending pointthan another skilled artisan based on research conditions, one of skillin the art understands that the hemagglutinin protein and the pB1 genemay be considered as a protein area or a gene area.

As used herein, a “Replikin sequence” is an amino acid sequence of 7 toabout 50 amino acids comprising or consisting of a Replikin motifwherein the Replikin motif comprises:

-   -   (1) at least one lysine residue located at a first terminus of        said peptide and at least one lysine residue or at least one        histidine residue located at a second terminus of said peptide;    -   (2) a first lysine residue located six to ten residues from a        second lysine residue;    -   (3) at least one histidine residue; and    -   (4) at least 6% lysine residues.        For the purpose of determining Replikin concentration, a        Replikin sequence must have a lysine residue at one terminus and        a lysine or a histidine residue at the other terminus. For        diagnostic, therapeutic, and preventive purposes, a Replikin        sequence may or may not have defined termini.

The term “Replikin sequence” can also refer to a nucleic acid sequenceencoding an amino acid sequence having 7 to about 50 amino acidscomprising:

-   -   (1) at least one lysine residue located six to ten amino acid        residues from a second lysine residue;    -   (2) at least one histidine residue; and    -   (3) at least 6% lysine residues,        wherein the amino acid sequence may comprise a terminal lysine        and may further comprise a terminal lysine or a terminal        histidine.

As used herein, the term “peptide” or “protein” refers to a compound oftwo or more amino acids in which the carboxyl group of one amino acid isattached to an amino group of another amino acid via a peptide bond.

As used herein, an “isolated” peptide may be synthesized by organicchemical methods. An isolated peptide may also be synthesized bybiosynthetic methods. An isolated peptide also may refer to a peptidethat is, after purification, substantially free of cellular material orother contaminating proteins or peptides from the cell or tissue sourcefrom which the peptide is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized by any method,or substantially free from contaminating peptides when synthesized byrecombinant gene techniques or a protein or peptide that has beenisolated in silico from nucleic acid or amino acid sequences that areavailable through public or private databases or sequence collections.An isolated peptide may be synthesized by biosynthetic or organicchemical methods.

Protein fragments, polypeptides, or peptides in this specification maybe chemically synthesized by any method known to one of skill in the artnow and hereafter. For example, isolated protein fragment, polypeptides,or peptides may be synthesized by solid phase synthesis. The productionof these materials by chemical synthesis avoids the inclusion of (or theneed to remove by purification) materials that are byproducts of otherproduction methods such as recombinant expression or isolation frombiological material. Such byproducts may include, for example, avianproteins associated with vaccines produced using birds' eggs orbacterial proteins associated with recombinant production in bacteria.

An “encoded” or “expressed” protein, protein sequence, protein fragmentsequence, or peptide sequence is a sequence encoded by a nucleic acidsequence that encodes the amino acids of the protein or peptide sequencewith any codon known to one of ordinary skill in the art now orhereafter. It should be noted that it is well-known in the art that, dueto redundancy in the genetic code, individual nucleotides can be readilyexchanged in a codon and still result in an identical amino acidsequence. As will be understood by one of ordinary skill in the art, amethod of identifying a Replikin amino acid sequence also encompasses amethod of identifying a nucleic acid sequence that encodes a Replikinamino acid sequence wherein the Replikin amino acid sequence is encodedby the identified nucleic acid sequence.

As used herein, “conserved” or “conservation” refers to conservation ofparticular amino acids due to lack of substitution. Conservation mayoccur at a specific position in a protein or polypeptide or may occur ata position that is close to a specific position in a protein orpolypeptide but not the exact specific position. This type ofconservation occurs because additional amino acid residues may besubstituted in a protein or polypeptide such that the numbering ofresidue positions may shift toward either terminus of the protein orpolypeptide.

As used herein, “Replikin Count” or “Replikin Concentration” refers tothe number of Replikin sequences per 100 amino acids in a protein,protein fragment, virus, or organism. A higher Replikin concentration ina first strain of a virus or organism has been found to correlate withmore rapid replication of the first virus or organism as compared to asecond, earlier-arising or later-arising strain of the virus or organismhaving a lower Replikin concentration. Replikin concentration isdetermined by counting the number of Replikin sequences in a givensequence, wherein a Replikin sequence is a peptide of 7 to about 50amino acid residues with a lysine residue on one end and a lysineresidue or a histidine residue on the other end wherein the peptidecomprises (1) a lysine residue six to ten residues from another lysineresidue, (2) a histidine residue, (3) and 6% or more lysine residues, orwherein a Replikin sequence is a nucleic acid that encodes a Replikinpeptide sequence.

Replikin Peptide Sequences Available for Therapies in Influenza VirusAcross Strains and Over Time

An aspect of the present invention provides compounds for diagnostic,therapeutic, and/or preventive purposes in influenza, methods ofdifferentiating infectivity and lethality in influenza, and methods ofdesigning therapies against influenza based on compounds of theinvention and differentiation of infectivity and lethality in influenza.

Compounds of the invention include Replikin peptides and homologues ofReplikin peptides identified in and isolated from different strains ofinfluenza and conserved over time in the same and different strains ofinfluenza. These Replikin peptides have been shown to be useful whencomprised in immunogenic compounds and have provided a protective effectagainst influenza infection including antagonism of both the infectivityof strains of influenza and the replication and lethality of strains ofinfluenza. Because these Replikin peptides are conserved within strainsof influenza over time and across different strains of influenza atconserved positions in the different strains of influenza, the ordinaryskilled artisan expects the functionality of these peptides to sharecommonality among various strains of influenza and among variousisolates of the same strain of influenza at different times.

Twelve peptides provided in an aspect of this invention were firstidentified as conserved in low-pathogenic H5N1 and high-pathogenic H5N1and were combined in a successful vaccine in chickens where infectivity,replication, and excretion of low-pathogenic H5N1 were all antagonizedor blocked by the vaccine. An exact homologue of one of the twelvepeptides was later identified as conserved at position 184 in isolatesof H1N1, high-pathogenic H5N1, and H9N2. See SEQ ID NO(s): 8 and 19.Further homologues were then identified in other isolates of H1N1 andH5N1. See SEQ ID NO(s): 13 and 20. Each of the homologues was positionedin the pB1 gene area of the virus. Based on the data presented hereinconcerning the function of the pB1 gene area in lethality in variousinfluenza viruses over time and the commonality and conservation of thehomologues, the applicants recognized that any of the homologues wouldbe useful as an immunogenic compound against any of the strains ofinfluenza virus in which a homologue had been or would be identified. Asa result, the applicants have developed methods of identifying otherhomologues of the twelve peptides contained in the successful vaccineagainst low-pathogenic H5N1. These homologues are now available for usein an immunogenic compounds that may be used against any strain ofinfluenza virus in which a homologue of one of the twelve peptides isidentified. They are further available against strains of influenzavirus where the homologues are present in the hemagglutinin or pB1 geneareas. In one aspect of the invention, a homologue may be 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more or 100% homologous with a peptideagainst which the homologue is compared. The methods have providedpeptides for a vaccine that may be applied for prevention or treatmentof any strain of influenza virus. The vaccine is known as TransFlu™.

The applicants have now additionally developed another vaccine thatcomprises eight additional peptides identified in the hemagglutininprotein area and pB1 gene area of the H1N1 virus. Homologues of any oneof these eight peptides may also be used in an immunogenic compoundagainst any strain of influenza virus that contains a homologue of oneof the eight peptides. A homologue of one these eight peptides maylikewise be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more or 100%homologous with a peptide against which the homologue is compared.

Because the peptides disclosed in the vaccines herein described arepeptides that are conserved over time in specific strains and sharedbetween strains (also over time), one of skill in the art expects suchpeptides (and peptides that are similar in structure and function) toalso be useful in immunogenic compounds for influenza infections ofvarious strains. This expectation is based on, for example, the functionof the peptides identified herein and the commonality of structure andposition of the peptides and their homologues as described herein aswell as the functionality of the peptides and the homologues in thehemagglutinin protein area and pB1 gene area in different strains ofinfluenza. See, e.g., FIGS. 2-5. This expectation is also based in parton the conservation of Replikin peptides generally and the commonalityof function of Replikin peptides across strains of influenza and acrossdifferent viruses and organisms. See, e.g., Tables 7a, 8, 9, and 10 withdescriptions and Examples 6 and 7 in U.S. application Ser. No.11/355,120, filed Feb. 16, 2006 and Table 8 with description in U.S.Pat. No. 7,442,761, and FIGS. 1-21 in U.S. application Ser. No.12/010,027, filed Jan. 18, 2008. For example, Replikin peptides havebeen shown to be broadly antigenic, to be conserved, and to be relatedto rapid replication and outbreaks across many different strains ofinfluenza virus. See, e.g., U.S. application Ser. No. 11/355,120, filedFeb. 16, 2006. Additionally, the crucial lysine and histidine residuesof Replikin peptides have been demonstrated to be related to rapidreplication and to be conserved in fixed positions within functionalproteins even in highly mutable viruses such as HIV. See, e.g., Table 8with description in columns 62 and 63 in U.S. Pat. No. 7,442,761.Further, as described herein the Replikin peptides and homologuesdisclosed herein are shown to be structurally and functionally relatedto the infectivity and lethality of influenza virus based on thepositions in the hemagglutinin protein area or pB1 gene area ofinfluenza, respectively. As a result, the peptides and their homologuesdescribed herein are, among other things, antigenic, common to variousstrains of influenza virus in both position and function, conserved invarious strains of influenza over time, conserved in specific positionsin the hemagglutinin protein area and pB1 gene areas over time,conserved in their lysines and histidines within the Replikin structure,and associated with mechanisms of infectivity and/or lethality. As aresult, one of ordinary skill in the art would expect the Replikinpeptides and their homologues described herein to be useful inimmunogenic compounds for therapies against influenza virus withinstrains, across strains, and across time.

Shared and Conserved Replikin Peptide Sequences and their Homologues

Replikins sequences and their homologues provided by an aspect of theinvention may be identified in strains of influenza virus including anystrain of influenza virus known now or identified or known hereafter.Compounds of the invention may be conserved within strains of influenzavirus, across types within strains of influenza virus, and acrossstrains of influenza virus. The compounds, because they are Replikinsequences, related to Replikin sequences, derived from Replikinsequences, identified as comprising Replikin sequences, or designed tocomprise Replikin sequences, are related to rapid replication,virulence, and lethality in influenza. See FIGS. 2-5. Compounds of theinvention, including conserved Replikin peptides, are useful asimmunogenic compounds to stimulate the immune system of a subject toproduce an immune response, which may include production of antibodiesor other binding molecules. Compounds of the invention are also usefulin therapies such as vaccines. Compounds of the invention are likewiseuseful in producing antibodies, antibody fragments, or other binding orantagonizing agents, which may be used, among other things, fordiagnostic and therapeutic purposes, including passive immunity.

The immunogenic compounds, antibodies (and other binding or antagonizingagents) and vaccines of the invention are useful against any strain ofinfluenza virus including influenza A, B, or C strains. Within strainsof influenza A, they are useful against any strain of influenza Aincluding, but not limited to, H1N1, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2,H7N7, H7N2, H7N3, H9N2, and H10N7. They are useful in any organism thatis capable of producing an immune response. The compounds of theinvention are also useful for diagnostic purposes, including identifyingrapidly replicating, virulent, or lethal strains of virus.

The compounds of the invention may be conserved in the H5N1 strain ofvirus including low-pathogenic (Low-Path) strains of H5N1 andhigh-pathogenic (High-Path) strains of H5N1. The compounds may also beconserved in other strains of influenza including H1N1, H1N2, H2N2,H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, and H10N7. For example,the following twenty-eight peptides and homologues of the followingtwenty-eight peptides are provided as an aspect of the invention asisolated or synthesized peptides, as immunogenic compounds, as vaccines,and as targets for antibodies and binding agents of the invention, amongother things: HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO:1),KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4),HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO:6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8),KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10),HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11),HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12), HFQRKRRVRDNVTK (SEQID NO: 13), HCQKTMNQVVMPK (SEQ ID NO: 14), HYQKTMNQVVMPK (SEQ ID NO:15), KRWRLFSKH (SEQ ID NO: 16), KKKHKLDK (SEQ ID NO: 17), KKKQRLTKX_(n)H(SEQ ID NO: 18) (where n=any amino acid from 1 to 41 residues),HFQRKRRVRDNMTK (SEQ ID NO: 19), HFQRKRRVRDNMTKKMVTQRTIGKKKQRLNK (SEQ IDNO: 20), KKGSSYPKLSKSYVNNKGKEVLVLWGVHH (SEQ ID NO: 21), HPVTIGECPKYVRSTK(SEQ ID NO: 22), KFEIFPKTSSWPNH (SEQ ID NO: 23), HNGKLCKLKGIAPLQLGK (SEQID NO: 24), KSYVNNKGKEVLVLWGVHH (SEQ ID NO: 25), KMNTQFTAVGKEFNH (SEQ IDNO: 26), KSQLKNNAKEIGNGCFEFYH (SEQ ID NO: 27), KHSNGTVK (SEQ ID NO: 28).

SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 are sequences that wereinitially identified in H1N1 or H5N1 as related to infectivity orlethality in those strains of influenza virus. Further investigation ofthe conservation of certain of those sequences in other strains ofinfluenza virus provided identical sequences or homologues of thosesequences conserved in other strains of influenza virus including H9N2,H3N2, H5N1, and H1N1 where the conserved homologues shared the sameamino acid residue position in the functional protein of the otherstrain of influenza virus. As a result, the conserved homologues wouldbe expected to share the same functional characteristics in those otherinfluenza viruses where they are conserved.

The conserved homologues are further identified in positions in thehemagglutinin protein area and pB1 gene areas of various strains ofinfluenza where these genes are directly associated with infectivity andlethality, respectively. Further, a vaccine based on these homologueshas provided successful results in chickens in antagonizing both theinfectivity of influenza virus and the replication (or lethality) ofinfluenza virus once it has entered a host system. See, e.g., Example 1below.

Information on the conservation of homologous sequences across variousstrains of influenza virus, therefore, provides sequences that offerimmunogenic compounds for antagonism of all strains comprising thesehomologues. As a result, a vaccine is provided herein (known asTransFlu™) that offers cross-strain protection for a variety of strainsof influenza.

For example, SEQ ID NO(s): 1-12 were initially identified in a strain ofLow-Path H5N1. These peptides have since that time been identified in aseries of highly pathogenic (High-Path) strains of H5N1 influenza,including a lethal strain of H5N1 isolated in Vietnam, among others.These peptides have been shown to provide a protective effect againstinfectivity and replication in host systems. SEQ ID NO(s): 13-20 havenow also been identified and isolated as homologues of at least oneamino acid sequence of SEQ ID NO(s): 1-12. Certain of these homologueshave been identified not only in strains of H5N1 but also in otherstrains of influenza virus such as H5N2, H3N2, and H1N1. Additionally,SEQ ID NO(s): 21-28 are also provided as a vaccine against H1N1.Homologues of these sequences in other strains of influenza are expectedto provide cross-strain protection.

Replikin peptides in general are seen to be conserved across strains ofinfluenza. In particular, amino acid residues that provide for theReplikin sequence structure of the peptides of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 is conserved widely across strains and time ininfluenza. The key amino acid residues that provide for the Replikinsequence structure are the lysine and histidine residues wherein aReplikin sequence has at least one lysine on one terminus and at leastone lysine or one histidine on the other terminus, at least one lysinethat is six to ten residues from at least one other lysine, at least onehistidine, and at least six percent lysines in total between theterminal lysine and the terminal lysine or histidine. Homologues of theReplikin peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 (wherethe lysines and histidines that create the Replikin structure areconserved) have been seen to be conserved widely across strains ofinfluenza virus.

As may be seen in FIG. 21 of U.S. application Ser. No. 11/1355,120,filed Feb. 16, 2006, when conserved homologues Replikin sequences arealigned one on top of the other over time, it is most apparent thatfixed and conserved portions of the structure of Replikin sequencesalign in a series of posts or girders that illustrate, like thestructure of a building, how key conserved amino acids provide constancyfor the survival of influenza over time as it mutates to avoid immunerecognition in its prospective host but maintains key functional geneticstructures that provide for continued replication of the virus. Thesekey functional genetic structures provide targets that Replikin-basedtherapies antagonize.

Compounds and Compositions Comprising Peptides Homologous to InfluenzaReplikin Peptides

One aspect of the present invention provides a protein, a proteinfragment, a polypeptide, or a peptide that comprises at least onepeptide A homologous with at least one peptide of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66. Peptide A may be 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% or more homologous or 100% homologous with any of thepeptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. The protein,protein fragment, or peptide may likewise be a peptide that consists ofa peptide A that is homologous with any of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66. A peptide consisting of any one of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 is also provided.

The amino acid sequence of the provided isolated or synthesized protein,protein fragment, polypeptide, or peptide may partially match an aminoacid sequence of an expressed whole protein. At least one, five, ten,twenty, thirty, forty, fifty, one hundred, two hundred, three hundred,four hundred, five hundred, five hundred and fifty or more amino acidresidues of the amino acid sequence of the expressed whole protein maynot be present in the protein, protein fragment, polypeptide, orpeptide. The amino acid sequence of the isolated or synthesized protein,protein fragment, polypeptide, or peptide may also partially match theamino acid sequence of an expressed whole protein where at least one,ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, onehundred, one hundred fifty, two hundred, two hundred fifty, threehundred, three hundred fifty, four hundred, four hundred fifty, fivehundred, five hundred fifty or more amino acid residues of at least oneterminus of the amino acid sequence of the expressed whole proteinis(are) not present at at least one terminus of said protein fragment,polypeptide, or peptide. Any additional number of amino acids may besituated on one or the other terminus or on both termini of the protein,protein fragment, polypeptide, or peptide.

Because a Replikin peptide, such as SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66, is associated with rapid replication, infectivity, and/orlethality, in functional proteins in influenza viruses, inclusion of anyReplikin peptide in a protein, protein fragment, polypeptide, or peptidedoes not negate the functional nature of the Replikin peptide. As such,antagonism of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 or a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66(with homology of 30% or greater) within a protein, protein fragment,polypeptide, or peptide would be expected to antagonize the replication,infectivity, and/or lethality of the protein, protein fragment,polypeptide, or peptide.

A provided peptide may further be a peptide B of 7 to about 50 aminoacid residues where peptide B contains a peptide A that is 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% or more homologous or 100% homologouswith any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. Anon-limiting peptide may further be a peptide A that is a Replikinpeptide wherein the Replikin peptide has a lysine residue on one end anda lysine residue or a histidine residue on the other end wherein theReplikin peptide comprises: (1) a lysine residue six to ten amino acidsfrom another lysine residue; (2) at least one histidine residue; and (3)at least 6% lysine residues.

An isolated or synthesized protein, protein fragment, polypeptide, orpeptide may consist of a peptide that is 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 100% homologous with at least one of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 where the length of the peptide is no more thanone, five, ten, twenty, thirty, forty, or fifty amino acid residueslonger than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66with which it is homologous. An isolated or synthesized proteinfragment, polypeptide, or peptide may likewise be no more than one, two,three, four, five, six, seven, eight, nine, or ten amino acid residueslonger than the sequence of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66with which it is homologous. An isolated or synthesized proteinfragment, polypeptide, or peptide may likewise consist of any one of thepeptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

An isolated or synthesized polypeptide or peptide may comprise a peptideA that has about the same number of amino acid residues as a peptide B,where peptide B is one of the peptides of SEQ ID NO: 1-28 and where thelysine residues and histidine residues in peptide A are conserved ascompared to the lysine residues and histidine residues in peptide B. Anisolated or synthesized polypeptide or peptide comprising peptide A mayhave up to 100 additional amino acid residues as compared to peptide B.Some or all of the up to 100 additional amino acid residues may bepositioned toward the amino-terminus and/or carboxy-terminus of thelysine or histidine termini of peptide A. Some of the additional aminoacid residues may be positioned within the lysine or histidine terminiof peptide A so long as a level of homology is maintained betweenpeptide A and peptide B that retains at least some of the functionalityof the Replikin peptide of peptide B. Functionality may include, but isnot limited to, antigenicity, rate of replication, antagonizability of aprotein containing said peptide A or said peptide B, binding capacity ofbinding agents to peptides A or B, etc.

An isolated or synthesized polypeptide or peptide may also comprise upto about 90, about 80, about 70, about 60, about 50, about 40, about 30,about 20, about 10, about 5, about 4, about 3, about 2, or about 1additional amino acid residues. The residues may be entirely outside ofthe Replikin structure or entirely within the Replikin structure orpartially within and partially outside the Replikin structure. A levelof homology should be maintained between peptides B and A whenadditional residues are present or are added. Residues outside of theReplikin structure are those residues on the amino-terminus orcarboxy-terminus of the polypeptide or peptide as compared to the lysineor histidine termini of peptide A. Residues within the Replikinstructure are those residues that are between the lysine or histidinetermini of peptide A. An isolated or synthesized polypeptide or peptidemay also consist of peptide A and peptide A may consist of peptide B.

An isolated or synthesized peptide may consist of a peptide of about 26amino acid residues with a histidine residue within zero, one, two,three, four, or five residues of the amino-terminus of the peptidewherein the histidine residue is considered to reside at position 1, andwherein relative to position 1 there is a lysine residue at position 8,a histidine residue at position 10, a lysine residue at position 13, alysine residue at position 18, and a lysine residue at position 26, andwherein up to one, two, three, four, or five additional residues may bepresent on the carboxy-terminus of the peptide after the lysine residueat position 26. If five residues are present on the amino-terminus ofposition 1 and five residues are present on the carboxy-terminus ofposition 26, the isolated or synthesized peptide will consist of about36 amino acids. Such an isolated or synthesized peptide is a homologueof SEQ ID NO: 1 and may be used as an immunogenic compound or as acomponent of a vaccine against infectivity in any strain of influenzavirus.

An isolated or synthesized peptide may consist of about 19 amino acidresidues with a lysine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the lysineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a histidine residue at position 3, a lysine residueat position 6, a lysine residue at position 11, and a lysine residue atposition 19, and wherein up to one, two, three, four, or five additionalresidues may be present on the carboxy-terminus of the peptide after thelysine residue at position 19. If five residues are present on each endof the peptide, it will consist of about 29 amino acids. Such anisolated or synthesized peptide is a homologue of SEQ ID NO: 2 and maybe used as an immunogenic compound or as a component of a vaccineagainst infectivity in any strain of influenza virus.

An isolated or synthesized peptide may consist of about 29 amino acidsresidues with a lysine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the lysineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 2, a lysine residue atposition 10, a histidine residue at position 28, and a histidine residueat position 29, and wherein up to one, two, three, four, or fiveadditional residues may be present on the carboxy-terminus of thepeptide after the histidine residue at position 29. If five residues arepresent on each end of the peptide, it will consist of about 39 aminoacids. Such an isolated or synthesized peptide is a homologue of SEQ IDNO: 3 and may be used as an immunogenic compound or as a component of avaccine against infectivity in any strain of influenza virus.

An isolated or synthesized peptide may consist of about 27 amino acidresidues with a histidine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the histidineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a histidine residue at position 2, a lysine residueat position 14, a lysine residue at position 19, and a lysine residue atposition 27, and wherein up to one, two, three, four, or five additionalresidues may be present on the carboxy-terminus of the peptide after thelysine residue at position 27. If five residues are present on each endof the peptide, it will consist of about 37 amino acids. Such anisolated or synthesized peptide is a homologue of SEQ ID NO: 4 and maybe used as an immunogenic compound or as a component of a vaccineagainst infectivity in any strain of influenza virus.

An isolated or synthesized peptide may consist of about 21 amino acidresidues with a histidine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the histidineresidue is considered to reside at position 1 and wherein relative toposition 1 there is a lysine residue at position 6, a lysine residue atposition 11, and a lysine residue at position 21, and wherein up to one,two, three, four, or five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 21.If five residues are present on each end of the peptide, it will consistof about 31 amino acids. Such an isolated or synthesized peptide is ahomologue of SEQ ID NO: 5 and may be used as an immunogenic compound oras a component of a vaccine against infectivity in any strain ofinfluenza virus.

An isolated or synthesized peptide may consist of about 22 amino acidresidues with a lysine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the lysineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 11, and a histidineresidue at position 22, and wherein up to one, two, three, four, or fiveadditional residues may be present on the carboxy-terminus of thepeptide after the histidine residue at position 22. If five residues arepresent on each end of the peptide, it will consist of about 32 aminoacids. Such an isolated or synthesized peptide is a homologue of SEQ IDNO: 6 and may be used as an immunogenic compound or as a component of avaccine against infectivity in any strain of influenza virus.

An isolated or synthesized peptide may consist of about 17 amino acidswith a lysine residue within zero, one, two, three, four, or fiveresidues of the amino-terminus of the peptide wherein the lysine residueis considered to reside at position 1, and wherein relative to position1 there is a lysine residue at position 9, and a histidine residue atposition 17, and wherein up to one, two, three four, or five additionalresidues may be present on the carboxy-terminus of the peptide after thehistidine residue at position 17. If five residues are present on eachend of the peptide, it will consist of about 27 amino acids. Such anisolated or synthesized peptide is a homologue of SEQ ID NO: 7 and maybe used as an immunogenic compound or as a component of a vaccineagainst lethality in any strain of influenza virus.

An isolated or synthesized peptide may consist of about 15 amino acidresidues with a histidine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the histidineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 5, a lysine residue atposition 14, and a lysine residue at position 15, and wherein up to one,two, three, four, or five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 15.If five residues are present on each end of the peptide, it will consistof about 25 amino acids. Such an isolated or synthesized peptide is ahomologue of SEQ ID NO: 8 and may be used as an immunogenic compound oras a component of a vaccine against lethality in any strain of influenzavirus.

An isolated or synthesized peptide may consist of about 18 amino acidresidues with a lysine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the lysineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 2, a histidine residueat position 5, a lysine residue at position 6, a lysine residue atpositions 11, 12, and 13, and a lysine residue at position 18, andwherein up to one, two, three, four, or five additional residues may bepresent on the carboxy-terminus of the peptide after the lysine residueat position 18. If five residues are present on each end of the peptide,it will consist of about 28 amino acids. Such an isolated or synthesizedpeptide is a homologue of SEQ ID NO: 9 and may be used as an immunogeniccompound or as a component of a vaccine against lethality in any strainof influenza virus.

An isolated or synthesized peptide may consist of about 14 amino acidresidues with a histidine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the histidineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 2, a lysine residue atpositions 7, 8, and 9, and a lysine residue at position 14, and whereinup to one, two, three, four, or five additional residues may be presenton the carboxy-terminus of the peptide after the lysine residue atposition 14. If five residues are present on each end of the peptide, itwill consist of about 24 amino acids. Such an isolated or synthesizedpeptide is a homologue of SEQ ID NO: 10 and may be used as animmunogenic compound or as a component of a vaccine against lethality inany strain of influenza virus.

An isolated or synthesized peptide may consist of about 26 amino acidresidues with a histidine residue within zero, one, two, three, four, orfive residues of the amino-terminus of the peptide wherein the histidineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 16, and a lysineresidue at positions 24, 25, and 26, and wherein up to one, two, three,four, or five additional residues may be present on the carboxy-terminusof the peptide after the lysine residue at position 26. If five residuesare present on each end of the peptide, it will consist of about 36amino acids. Such an isolated or synthesized peptide is a homologue ofSEQ ID NO: 11 and may be used as an immunogenic compound or as acomponent of a vaccine against lethality in any strain of influenzavirus.

An isolated or synthesized peptide may consist of about 35 amino acidresidues with a histidine residue within zero, one, two, three, four, orfive residues of the amino terminus of the peptide wherein the histidineresidue is considered to reside at position 1, and wherein relative toposition 1 there is a lysine residue at position 6, a lysine residue atposition 28, and a lysine residue at position 35, and wherein up to one,two, three, four, or five additional residues may be present on thecarboxy-terminus of the peptide after the lysine residue at position 35.If five residues are present on each end of the peptide, it will consistof about 45 amino acids. Such an isolated or synthesized peptide is ahomologue of SEQ ID NO: 12 and may be used as an immunogenic compound oras a component of a vaccine against lethality in any strain of influenzavirus.

Any one of the above-listed isolated or synthesized peptides may have anamino-terminus at position 1 and a carboxy-terminus at the amino acidresidue for which a position is expressly numbered where thatexpressly-numbered position is the farthest numbered position toward thecarboxy-terminus of the peptide. For example, a homologue of SEQ ID NO:7 (KDVMESMDKEEMEITTH) will have a terminal lysine at position 1 and aterminal histidine at position 17 or a homologue of SEQ ID NO: 4(HHSNEQGSGYAADKESTQKAIDGITNK) will have a terminal histidine at positionnumber 1 and a terminal lysine at position number 27.

The at least one isolated or synthesized protein, protein fragment, orpeptide may also comprise at least one peptide A and at least onepeptide C where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% homologous with at least one peptide of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 and where peptide C is at least 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 100% homologous with at least one peptide ofSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. Peptide C may be homologouswith a different peptide from among SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66 than the peptide that peptide A is homologous with. The atleast one isolated or synthesized protein, protein fragment, or peptidemay comprise three or more peptides homologous with at least threedifferent peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

All of the above-discussed proteins, protein fragments, polypeptides,and peptides comprise the functional unit of a homologue of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66. These proteins, protein fragments,polypeptides, and peptides share a functional role in either infectivityor lethality. Antagonism of any of the homologues of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 will likewise antagonize either the infectivityfunction or lethality function in any strain of influenza that share ahomologue of any one of the sequences. As a result, the proteins,protein fragments, polypeptides, and peptides are useful as immunogeniccompounds, therapeutic compounds, vaccines, and for other therapiesdirected at antagonizing the infectivity and/or lethality of a strain ofinfluenza. When comprised in a vaccine, disclosed proteins, proteinfragments, polypeptides, and peptides are expected to be capable oflimiting the excretion or shedding of influenza virus such that thevirus is limited in its spread from host to host or from host toreservoir to host, etc. As such, disclosed compounds are effective atlimiting sources of influenza infection. Likewise, any binding agentthat binds one of the proteins, protein fragments, polypeptides, andpeptides discussed above will antagonize the infectivity and/orlethality of a strain of influenza and limit sources of influenzainfection such as transmission from host to host or from host toreservoir to host.

Immunogenic Compositions Comprising Peptide Homologous to InfluenzaReplikin Peptides

As such, a non-limited protein, protein fragment, or peptide of theinvention may be comprised in an immunogenic compound. The proteins,protein fragment, polypeptides, and peptides provided by an aspect ofthe invention comprise at least a portion that is homologous with aReplikin peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. Thesehomologues are expected by one of ordinary skill in the art to stimulatethe immune system of a subject upon sufficient exposure to produceantibodies against at least the homologous portion of the protein,protein fragment, polypeptide, or peptide. One of ordinary skill in theart would expect that antibodies or other binding agents arrayed againsta protein or protein fragment comprising one of the antigenic homologuesdisclosed herein would be antagonized.

One of ordinary skill would also expect an antagonist of one of thesehomologues to antagonize any influenza virus that comprises a homologuewithin its hemagglutinin protein area or pB1 gene area since an immuneresponse against SEQ ID NO(s): 1-12 has been shown to antagonize boththe infectivity and replication (including excretion) of H5N1. Becausehomologues of SEQ ID NO(s): 1-12 have been shown to be conserved acrossstrains of influenza in the hemagglutinin protein area and the pB1 genearea, one of ordinary skill would expect antagonism of such homologuesto result in antagonism of influenza replication similar to what wasobserved in SEQ ID NO(s): 1-12 in chickens. One of ordinary skill wouldfurther expect particular antagonism of the infectivity and lethalitymechanisms of influenza when an immune system is stimulated against ahomologue of SEQ ID NO(s): 1-6 and SEQ ID NO(s): 7-12, respectively.

As a result, the applicants disclose herein a series of homologues ofSEQ ID NO(s): 1-12 identified in the hemagglutinin and pB1 gene areas ofa wide range of strains of influenza. Each of these homologues isprovided as a component that may be used in an immunogenic compound tostimulate the immune system of a subject against influenza infection.Additionally, other homologous sequences are likewise provided asimmunogenic compounds to stimulate the immune system of a subjectagainst influenza infection. Any homologue that shares 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or more homology with any one of SEQ ID NO(s):1-12 is disclosed as a peptide that may be used in an immunogeniccompound against influenza infection. Additionally, any protein, proteinfragment, polypeptide, or peptide comprising such a homologue may beused as an immunogenic compound or be comprised within an immunogeniccompound. An immune response against such compounds would be understoodby one of ordinary skill in the art to be useful in stimulating theimmune system against an influenza infection.

Likewise, any homologue that shares 30%, 40%, 50%, 60%, 70%, 80%, 90%,95% or more homology with any one of SEQ ID NO(s): 21-28 is disclosed asa peptide that may be used in an immunogenic compound against influenzainfection. Any protein, protein fragment, polypeptide, or peptidecomprising such a homologue may also be used as an immunogenic compoundor may be comprised within an immunogenic compound. An immune responseagainst such compounds would be understood by one of ordinary skill inthe art to be useful in stimulating the immune system against aninfluenza infection.

Vaccines Comprising Peptides Homologous to Influenza Replikin Peptides

An immunogenic compound provided as an aspect of the invention may beused as a component of a non-limiting vaccine against any strain ofinfluenza. A vaccine comprising one or more homologues of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66 may be used against influenza infection.Likewise, a vaccine comprising one or more homologues of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66 derived from a hemagglutinin protein areaand one or more homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 derived from a pB1 gene area may be used against influenzainfection and may antagonize the infectivity and/or replication andlethality of an influenza infection. Further, mixtures of homologues ofSEQ ID NO(s): 1-6 and SEQ ID NO(s): 7-12 are provided as vaccines toantagonize both the infectivity and replication and lethality of aninfluenza infection. Such vaccines are useful for antagonizinginfectivity, replication, lethality, and excretion or spread ofinfluenza virus.

A non-limiting vaccine is provided comprising: at least one proteinfragment, polypeptide, or peptide comprising at least one peptide A,where peptide A is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or100%, homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66 and wherein peptide A is isolated from a hemagglutinin proteinarea of influenza virus, or a synthesized version thereof; and at leastone protein fragment, polypeptide, or peptide comprising at least onepeptide B, where peptide B is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%or 95%, or 100%, homologous with at least one of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 and wherein peptide B is isolated from a proteinor peptide encoded by a pB1 gene area of influenza virus, or asynthesized version thereof. The peptide A may be identified in,isolated from, derived from, or synthesized from an isolate of influenzavirus predicted to have a greater infectivity than at least one otherisolate of influenza virus and the peptide B may be identified in,isolated from, derived from, or synthesized from an isolate of influenzavirus predicted to have a greater lethality than at least one otherisolate of influenza virus. A vaccine may further comprise a pluralityof protein fragments, polypeptides, and/or peptides from thehemagglutinin protein area and a plurality of protein fragments,polypeptides, and/or peptides from the pB1 gene area.

A vaccine may further comprise at least one Replikin peptide from thehemagglutinin protein area and at least one Replikin peptide from thepB1 gene area. A vaccine may further comprise a plurality of Replikinpeptides from a hemagglutinin protein area where the at least oneReplikin peptide from a pB1 gene area is a plurality of Replikinpeptides from a pB1 gene area. A vaccine may comprise a plurality of theshortest Replikin peptides from a hemagglutinin protein area and aplurality of the shortest Replikin peptides from a pB1 area. A vaccinemay comprise the shortest Replikin peptides from a hemagglutinin proteinarea identified in an influenza virus isolate or a plurality ofinfluenza virus isolates predicted to have a greater infectivity than atleast one other isolate of influenza virus and may comprise the shortestReplikin peptides from a pB1 gene area identified in an influenza virusisolate or a plurality of influenza virus isolates predicted to have agreater lethality than at least one other isolate of influenza virus.

A vaccine may further comprise a plurality of the longest Replikinpeptides from a hemagglutinin protein area and a plurality of thelongest Replikin peptides from a pB1 area. A vaccine may comprise thelongest Replikin peptides from a hemagglutinin protein area identifiedin an influenza virus isolate or a plurality of influenza virus isolatespredicted to have a greater infectivity than at least one other isolateof influenza virus and may comprise the longest Replikin peptides from apB1 gene area identified in an influenza virus isolate or a plurality ofinfluenza virus isolates predicted to have a greater lethality than atleast one other isolate of influenza virus. A vaccine may also comprisea mixture of the shortest and longest Replikin peptides in thehemagglutinin protein area and/or pB1 gene area.

A vaccine may be directed against any influenza virus including,influenza A, influenza B, or influenza C. A vaccine may be directedagainst H1N1, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3,H9N2, H10N7, or any other strain of influenza A virus. Any of thesevaccines may be synthesized in seven days or less, which allows foradministration of vaccines that are a best fit for a particular virulentstrain of virus.

A vaccine may be formulated with a pharmaceutically acceptableexcipient, carrier, or adjuvant. One pharmaceutically acceptable carrieror excipient is water. Excipients, carriers, or adjuvants may include,but are not limited to, excipients, carriers and adjuvants known tothose of skill in the art now or hereafter.

The compositions of the invention may be formulated for delivery by anyavailable route including, but not limited to parenteral (e.g.,intravenous), intradermal, subcutaneous, oral, nasal, bronchial,ophthalmic, transdermal (topical), transmucosal or any other routes. Asused herein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forintranasal, intraocular, spray inhalation, parenteral (e.g.,intravenous), intramuscular, intradermal, or subcutaneous applicationcan include the following components: a sterile diluent such as water(for dermal, nasal, or ocular application, spraying, or injection),saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. Preparations may be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. Preferred pharmaceuticalformulations are stable under the conditions of manufacture and storageand must be preserved against the contaminating action of microorganismssuch as bacteria and fungi. In general, the relevant carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above.

Administration of the vaccine via any method may produce an immuneresponse in the animal or human, it may further produce an antibodyresponse in the animal or human. In a further non-limiting embodiment,the vaccine may produce a protective effect in the animal or human. Forexample, the vaccine of the present invention may be administered to arabbit, a chicken, a shrimp, a pig, a ferret, a human, or any animalcapable of an immune response. Because of the universal nature ofReplikin sequences, a vaccine of the invention may be directed at arange of strains of influenza.

The vaccines of the present invention can be administered alone or incombination with antiviral drugs, such as gancyclovir; interferon;interleukin; M2 inhibitors, such as, amantadine, rimantadine;neuraminidase inhibitors, such as zanamivir and oseltamivir; and thelike, as well as with combinations of antiviral drugs.

Generally, the dosage of peptides is in the range of from about 0.01 μgto about 500 mg, from about 0.05 μg to about 200 mg, about 0.075 μg toabout 30 mg, about 0.09 μg to about 20 mg, about 0.1 μg to about 10 mg,about 10 μg to about 1 mg, and about 50 μg to about 500 μg. The skilledpractitioner can readily determine the dosage and number of dosagesneeded to produce an effective immune response.

Compositions Comprising any of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66

A non-limiting composition is provided comprising one or more isolatedor synthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more homologous with at least one of the peptides of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66. A composition is likewise providedcomprising one or more isolated or synthesized peptides that are 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with at least oneof the peptides of SEQ ID NO(s): 1-12 or SEQ ID NO(s): 21-28. Acomposition is provided comprising one or more isolated or synthesizedpeptides consisting of at least one peptide of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 or at least one peptide of SEQ ID NO(s): 1-12 orat least one peptide of SEQ ID NO(s): 21-28. A composition is furtherprovided comprising two, three, four five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, or twenty or more isolated or synthesized peptidesof SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

A composition comprising a mixture of peptides is provided wherein themixture comprises at least each of the isolated or synthesized peptidesof SEQ ID NO(s): 1-12 and/or at least each of the isolated orsynthesized peptides of SEQ ID NO(s): 21-28. A mixture is provided thatis equimolar. A mixture is also provided that is equal by weight. Such acomposition may comprise about 10% by weight the peptide of SEQ ID NO:1, it may comprise about 9% by weight the peptide of SEQ ID NO: 2, itmay comprise about 10% by weight the peptide of SEQ ID NO: 3, it maycomprise about 6% by weight the peptide of SEQ ID NO: 4, it may compriseabout 8% by weight the peptide of SEQ ID NO: 5, it may comprise about 8%by weight the peptide of SEQ ID NO: 6, it may comprise about 7% byweight the peptide of SEQ ID NO: 7, it may comprise about 6% by weightthe peptide of SEQ ID NO: 8, it may comprise about 10% by weight thepeptide of SEQ ID NO: 9, it may comprises about 8% by weight the peptideof SEQ ID NO: 10, it may comprise about 7% by weight the peptide of SEQID NO: 11, and/or it may comprise about 11% by weight the peptide of SEQID NO: 12.

The composition may further comprise two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, or more isolated or synthesizedpeptides of SEQ ID NO(s): 1-12 or two, three, four, five, six, seven,eight, or more isolated or synthesized peptides of SEQ ID NO(s): 21-28.The composition may also comprise any number of peptides of SEQ IDNO(s): 13-20. The composition may also comprise one or more isolated orsynthesized peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%or more homologous with at least one of peptides SEQ ID NO(s): 1-12, SEQID NO(s) 13-20, and/or SEQ ID NO(s): 21-28. The composition may comprisetwo, three, four, five, six, seven, eight, nine, ten, eleven, twelve ormore isolated or synthesized peptides that are 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95% or more homologous with at least one of peptides SEQ IDNO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s): 21-28. Thecomposition may further comprise a mixture of peptides comprisingisolated or synthesized peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s):13-20, or SEQ ID NO(s): 21-28.

Conserved Replikin Peptides Across Influenza Strains

Identification of conserved Replikin peptides across strains ofinfluenza virus has provided for the development of vaccines that may bedirected across strains of influenza virus. Identification of conservedReplikin peptides in isolates of influenza of any strain may beaccomplished in any way known to one of skill in the art now orhereafter. One method is by review of in silico sequences provided atwww.pubmed.com. Peptides that share exact identity or 100% homology withearlier identified Replikin peptides may be tracked using computersearching methods. Peptides that share 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% or more homology with an earlier identified Replikin peptidemay also be tracked by computer methods.

For example, a vaccine has now been developed for prevention andtreatment of infection of H5N1 virus. See, e.g., Example 1 below. Thesequences that are used in the vaccine in Example 1 have now beenidentified as conserved across many different strains. For example, SEQID NO: 8 (HFQRKRRVRDNMTKK), which was originally identified in the pB1gene area of H5N1, shares homology with SEQ ID NO: 13 (HFQRKRRVRDNVTK),which has been identified as conserved in the pB1-F2 gene area of H1N1.SEQ ID NO: 8 is homologous with SEQ ID NO: 13 in that the valine atposition 12 in SEQ ID NO: 13 is substituted with a methionine in SEQ IDNO: 8. SEQ ID NO: 8 also has one additional lysine on its C-terminus. Asa result of this homology, a vaccine comprising SEQ ID NO: 8 or SEQ IDNO: 13 may be used against either H1N1 or H5N1 or any other strainexpressing a homologue of these sequences. If such a homologue isexpressed in the pB1 gene area or the pB1-F2 gene area of a strain, thevaccine will be particularly useful against such a strain. Further, avaccine containing SEQ ID NO(s): 1-12, as described above, is availableas a vaccine against H1N1 strains as well as H5N1 strains of influenzavirus since such a vaccine comprises the peptide of SEQ ID NO: 8.

Sequences that are homologues of SEQ ID NO(s): 1-12 are appropriatesequences for inclusion in a vaccine directed against influenza virusincluding, H1N1, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3,H9N2, H10N7, or any other strain of influenza A virus and against anystrain of influenza B or influenza C virus. Likewise, sequences that arehomologues of SEQ ID NO(s): 13-20 are appropriate sequences forinclusion in a vaccine directed against influenza virus including, H1N1,H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, orany other strain of influenza A virus and against any strain ofinfluenza B or influenza C virus. Sequences that are homologues of SEQID NO(s): 21-28 are also appropriate sequences for inclusion in avaccine directed against influenza virus including, H1N1, H1N2, H2N2,H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any otherstrain of influenza A virus and against any strain of influenza B orinfluenza C virus.

The above-discussed homologues are expected by one of ordinary skill inthe art to provide antigenicity that is comparable to any one of SEQ IDNO(s): 1-12, SEQ ID NO(s): 13-20, SEQ ID NO(s): 21-28. Further, becausethese homologues are often conserved in the hemagglutinin and pB1 geneareas of different strains of influenza virus, these homologues areuseful for developing antagonists against influenza infections,including for vaccinating a subject with the homologous peptides tostimulate the immune system of the subject against the peptides andin-turn against influenza virus proteins harboring such peptides orother homologues of such peptides.

Homology that is sufficient to produce a useful target for antagonismincludes peptides that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or upto 100% homologous with any of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66. Homology may be determined with peptides wherein gaps exists inthe sequence that is being compared to any one of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 between amino acids that are identical to thoseof the peptide chosen from SEQ ID NO(s): 1-12. For example, SEQ ID NO: 1(HAQDILEKEHNGKLCSLKGVRPLILK) would be considered more than 86%homologous with the following sequence HAQDILEKEHNGKLCSLKGVRPX_(n=4)LILK(SEQ ID NO: 29) because SEQ ID NO: 1 is identical to SEQ ID NO: 29 inall residues except for the four residues represented by X_(n=4).Likewise, SEQ ID NO: 1 is more than 86% homologous withHAQDXILEKEHNGKLCXSLKGVRXXPLILK (SEQ ID NO: 30) because it is identicalto SEQ ID NO: 30 in all residues except for the residues represented byX. Because SEQ ID NO(s) 29 and 30 are 86% homologous with SEQ ID NO: 1,SEQ ID NO(s): 29 and 30 are available as peptides for inclusion in avaccine directed against infectivity in H5N1 or in any influenza virusstrain wherein homologues to SEQ ID NO: 1 are conserved.

Concerning gaps, the number of gaps in either the basis sequence or theidentified sequence should be limited to the number of gaps allowablewithout significantly compromising the function of the identifiedsequence as compared to the basis sequence. In general, many gaps in thesequence of the basis peptide or in the sequence of the identifiedpeptide are allowed based on homology as defined herein. Relatively moregaps are allowed if the lysines and histidines that create thedefinition of the Replikin peptide are identically shared between thebasis peptide and the identified peptide. Relatively more gaps are alsoallowed if the lysines and histidines that create the definition of theReplikin peptide are shared at least in close position (for examplewithin ten, nine, eight, seven, six, five, four, three, two, or oneamino acid residue). If some of the lysines and histidines that createthe definition of the Replikin peptide are not present in the identifiedpeptide, fewer gaps may be allowed. Nevertheless, if the identifiedpeptide functions similarly to the basis peptide, any number of gaps areallowed. In general, three or more gaps are allowed in the sequence ofthe basis peptide or in the sequence of the identified peptide withinten amino acid residues of the basis peptide if no lysines or histidinesare present in the identified peptide. Two or more gaps or one or moregaps are also allowed. Nevertheless, if the identified sequence providesthe same or a similar function to the basis sequence, more gaps areallowed up to the number of gaps that will provide a homology of 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or more homology. Additionally, wherethe lysines and histidines of the Replikin definition are present inboth the identified peptide and the basis peptide, there should be nolimit on how many gaps are allowed.

The presence of lysines and histidines providing for the Replikindefinition in an identified peptide requires significantly less homologybecause the lysines and the histidines of the Replikin definitionprovide for conservation of Replikin function. For example, in Table 8and the description thereof in columns 62 and 63 in U.S. Pat. No.7,442,761, a highly mutable tat protein in HIV is described andanalyzed. As may be seen from Table 8 in U.S. Pat. No. 7,442,761, in tatprotein of HIV, which is essential for replication in the virus, lysinesand histidines that are essential to maintaining the Replikin definitionwithin a key Replikin peptide in the protein are observed to be 100%conserved, while substitutions in amino acid residues that are notessential to maintaining the Replikin definition are commonlysubstituted. The conservation of the key amino acids for maintaining theReplikin definition is understood to provide a specific survivalfunction for HIV. The same phenomenon is seen in influenza. See U.S.Pat. No. 7,442,761, column 62, lines 42-45.

Sequences that are conserved across strains of influenza in thehemagglutinin and pB1 gene areas are excellent targets for controllinginfectivity and lethality, respectively. As such, identification ofconserved Replikin sequences in the hemagglutinin and pB1 gene areas indifferent strains of influenza including H1N1, H1N2, H2N2, H3N2, H3N8,H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any other strain ofinfluenza A virus and against any strain of influenza B or influenza Cvirus, provides sequences that are provided as included in across-strain vaccine for influenza virus.

Conserved Replikin Sequences in Hemagglutinin Protein and the pB1 GeneArea in Vaccines to Provide Cross-Strain Protection

One aspect of the invention, therefore, provides conserved sequences inthe hemagglutinin protein area and conserved sequences in the pB1 genearea for diagnostic, predictive, and therapeutic purposes, includingvaccines that provide cross-strain influenza protection. Replikinsequences that are shared across strains and Replikin sequencehomologues that are shared across strains provide targets fordiagnostic, predictive, and therapeutic purposes. Because Replikinsequences are associated by the applicants with mechanisms of rapidreplication and because Replikin sequences in the hemagglutinin proteinarea and the pB1 gene area are associated by the applicants withinfectivity and lethality, respectively, Replikin sequences that areshared across influenza strains or homologues of Replikin sequences thatare shared across influenza strains provide excellent targets fordiagnostics and therapeutics directed at these shared sequences.Identifying these targets provides for therapies such as a vaccine or abinding agent (e.g., an antibody or antibody fragment) that may bedirected at Replikin sequences or their homologues in an array ofinfluenza types and strains.

Replikin sequences have been identified by the applicants that areshared among, for example, H1N1, H5N1, H3N2, and H9N2. Such conservedsequences provide targets for vaccines that provide cross-strainprotection in these strains of influenza A and provide cross-strainprotection in strains that share the sequences or homologues of thesequences. For example, the applicants have identified five Replikinsequences in the H1N1 pB1-F2 gene area that are conserved within H1N1and are shared with H5N1 or H3N2.

One such sequence is HFQRKRRVRDNVTK (SEQ ID NO: 13). SEQ ID NO: 13 hasbeen observed to be conserved at position 184 in the pB1-F2 gene area inisolates of H1N1 since at least 1948 and shares homology with SEQ ID NO:8 (HFQRKRRVRDNMTKK), which was originally identified by the applicantsin the pB1 gene area of H5N1 and has been observed to be conserved from2000 through 2009 in isolates of H5N1 virus. See, e.g., Accession No.AAF74314. SEQ ID NO: 8 is homologous with SEQ ID NO: 13 in that thevaline at position 12 in SEQ ID NO: 13 is substituted with a methioninein SEQ ID NO: 8. SEQ ID NO: 8 also has one additional lysine on itsC-terminus. As a result of this homology, a vaccine comprising SEQ IDNO: 8 or SEQ ID NO: 13 may be used against either H1N1 or H5N1 or anyother strain expressing a homologue of these sequences. If such ahomologue is expressed in the pB1 gene area or the pB1-F2 gene area of astrain, the vaccine will be particularly useful against such a strain.Further, a vaccine containing SEQ ID NO(s): 1-12, as described above, isavailable as a vaccine against H1N1 strains as well as H5N1 strains ofinfluenza virus since such a vaccine comprises the peptide of SEQ ID NO:8. A vaccine comprising SEQ ID NO(s): 1-12, then, provides an example ofa vaccine to be used as a cross-strain vaccine.

As a further example of the conservation of Replikin sequences in thepB1-F2 gene area of various strains of influenza, the sequenceHCQKTMNQVVMPK (SEQ ID NO: 14) has been observed as conserved at position41 in the pB1-F2 gene area of isolates of H1N1 since 1918 and ahomologue is also conserved at position 41 in the pB1-F2 gene area ofisolates of H3N2 in at least 1968, 2004, 2006, and 2008. See, e.g.,Accession Nos. ABI922289, ACK99430, ACI26481, ACI26437, and ACI 26294.In addition, SEQ ID NO: 14 is further conserved in H1N1 with asubstitution of the cysteine residue at position 2 by a tyrosineresidue. The resulting sequence is HYQKTMNQVVMPK (SEQ ID NO: 15), whichhas been observed as conserved at position 41 in the pB1-F2 gene area ofH1N1 isolates of H1N1 from at least 1951 through 1983. SEQ ID NO: 15 hasalso been observed as conserved in H5N1. In view of the conservation ofSEQ ID NO(s): 14 and 15 or their homologues in H1N1, H5N1, and H3N2, avaccine comprising a peptide of SEQ ID NO(s): 14 or 15 or homologues ofone of those sequences is available as a vaccine against H1N1, H5N1, andH3N2 strains of influenza virus or any strain expressing a homologue ofthe peptides.

The sequence KRWRLFSKH (SEQ ID NO: 16) has been observed to be conservedat position 78 of the pB1-F2 gene area of H1N1 in isolates from 1918through 2008. SEQ ID NO: 16 is also conserved at position 78 of thepB1-F2 gene area of H3N2 in at least 1968 and 2008. See, e.g. AccessionNos. ABI92289, ACK99430, ACI26481, ACI26437, ACI26294. A vaccinecomprising SEQ ID NO: 16 is, therefore, available against both H1N1 andH3N2 or any other influenza strain expressing one or more homologues ofSEQ ID NO: 16.

The sequence KKKHKLDK (SEQ ID NO: 17) is also conserved at position 207of the pB1-F2 gene area of isolates of H1N1 in isolates from at least1991 through 2009. SEQ ID NO: 17 is also conserved in H5N1. A homologueof SEQ ID NO: 17, namely, sequence KKKQRLTKX_(n)H (SEQ ID NO: 18) (wheren=any amino acid from 1 to 41 residues), is conserved in the pB1 genearea of isolates of H1N1 and H5N1 at position 207. As such, a vaccinecomprising SEQ ID NO(s): 17 or 18 is available against H1N1 and H5N1 orany influenza strain expressing homologues of these sequences.

The sequence HFQRKRRVRDNMTK (SEQ ID NO: 19) is also conserved atposition 184 in the pB1 gene area of H5N1 in isolates from at least 2000through 2009. See, e.g., Accession No. AAF74314. SEQ ID NO: 19 is a 93%homologue with SEQ ID NO: 8 with only one additional lysine on thec-terminus. SEQ ID NO: 19 is also a homologue of SEQ ID NO: 13 withabout 93% homology. SEQ ID NO: 19 is conserved in H1N1 at position 184as well as in H9N2.

Another homologue of SEQ ID NO(s): 8 and 19 isHFQRKRRVRDNMTKKMVTQRTIGKKKQRLNK (SEQ ID NO: 20), which is conserved atposition 184 in the pB1 gene area of H5N1 isolates from at least 2000through 2005. See Accession No. AAF74314. SEQ ID NO: 20 is 48%homologous with SEQ ID NO: 8 and 45% homologous with SEQ ID NO: 19. Allof these sequences share homology with SEQ ID NO: 13, which has beenobserved to be conserved at position 184 in the pB1-F2 gene area inisolates of H1N1 since at least 1948. A vaccine comprising SEQ ID NO(s):8, 13, 19, or 20, or any combination thereof, is available against H1N1,H5N1, H9N2 or any influenza strain expressing homologues of thesesequences.

Methods of Designing Vaccines

The invention also provides methods of designing and making vaccines.For example, the invention provides a method of making a vaccinecomprising selecting at least one or more isolated or synthesizedpeptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQ ID NO(s):21-28 as a component of a vaccine and making said vaccine. The methodmay comprise selecting from 1 to up to 12 or more isolated orsynthesized peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s): 13-20, or SEQID NO(s): 21-28 as a component of a vaccine. The method may compriseidentifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s):13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virusup to about 3 years before the vaccine is made. The method may compriseidentifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s):13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virusup to about 1 year before the vaccine is made. The method may compriseidentifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s):13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virusup to about 6 months before the vaccine is made. The method may compriseidentifying one or more peptides of SEQ ID NO(s): 1-12, SEQ ID NO(s):13-20, or SEQ ID NO(s): 21-28 in an emerging strain of influenza virusup to about 7 days before the vaccine is made.

An emerging strain may be any strain of influenza virus identified byone of skill in the art as a strain of influenza virus that is predictedto expand in a population in hosts or that is predicted to increase invirulence, morbidity, and or mortality in its hosts. An emerging strainmay likewise be a strain of influenza virus wherein Replikinconcentration is observed to be increasing over time. An emerging strainmay likewise be a strain of influenza virus identified within a risingportion of Replikin cycle, following a peak in a Replikin cycle,following a step-wise rise in a Replikin cycle, or identified by aReplikin Count Virus Expansion Index as an emerging strain of virus. SeeU.S. application Ser. No. 12/429,044, filed Apr. 23, 2009, which isincorporated herein by reference.

A method of making a vaccine is also provided comprising: selecting atleast one isolated or synthesized protein, protein fragment,polypeptide, or peptide comprising a homologue of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 as a component of a vaccine; and making saidvaccine. An isolated or synthesized protein, protein fragment,polypeptide, or peptide may comprise a peptide that is 30%, 40%, 50%,60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQID NO(s): 1-12, 13-20, 21-28, and 32-66. At least two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, or more homologuesof SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 may be selected. Also, atleast two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, or more peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66may be selected. The isolated or synthesized protein, protein fragment,polypeptide, or peptide may have the same amino acid sequence as atleast one protein, protein fragment, polypeptide or peptide identifiedin an emerging strain of influenza virus up to one, two, or three ormore years prior to making said vaccine. The at least one protein,protein fragment, polypeptide or peptide may be identified in anemerging strain of influenza virus one week, one month, two months,three months, four months, five months, or six months prior to makingsaid vaccine.

A method of making a vaccine is provided comprising: selecting as acomponent of the vaccine at least one protein fragment, polypeptide, orpeptide comprising at least one peptide A, where peptide A is at least30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with atleast one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 isolated from ahemagglutinin protein area (or a synthesized version thereof), andselecting as a component of the vaccine at least one protein fragment,polypeptide, or peptide comprising at least one peptide B, where peptideB is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%,homologous with at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 isolated from a pB1 gene area (or a synthesized version thereof);and making a vaccine comprising the components.

A method of making a vaccine is also provided comprising: identifying(1) at least one protein, protein fragment, polypeptide, or peptide of ahemagglutinin protein area in or derived from an isolate of influenzavirus having relatively greater infectivity than another isolate ofinfluenza virus or a plurality of isolates of influenza viruses, and (2)at least one protein, protein fragment, polypeptide, or peptide of a pB1gene area in or derived from an isolate of influenza virus havingrelatively greater lethality than another isolate of influenza virus ora plurality of isolates of influenza virus; and making a vaccinecomprising the at least one protein, protein fragment, polypeptide, orpeptide of a hemagglutinin protein area and the at least one protein,protein fragment, polypeptide, or peptide of a pB1 gene area.

The invention also provides a kit for making a vaccine where the kitincludes at least one isolated or synthesized peptide of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66 or homologues of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66. The kit may also include two, three, four, andup to twelve or more peptides of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 or homologues of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

For the first time, new non-biological software and organic chemicaltotally synthetic methods have been developed to manufacture vaccines,based on the discovery of Replikins. The centrality of Replikins toinfluenza infectivity has been recently confirmed by the data of twogroups, Harvard-CDC and Scripps-Crucell, demonstrating that inhibitoryantibody lands on and binds selectively to previously defined Replikins.Three months to one year in advance of any outbreak, the relatedFluForecast® software technology warns of the coming emergent disease,as recently demonstrated one year in advance of the current H1N1outbreak, and defines the Replikins to be synthesized in the vaccine.

The new vaccine technology has been tested and demonstrated to work inindependent trials against influenza H5N1 virus in chickens, and againstlethal Taura Syndrome virus in shrimp. Both TransFlu™ (the firstsynthetic cross-strain Pan Flu vaccine) and Taura Syndrome Virusvaccines have been manufactured in 7 days. Kilogram amounts of thesevaccines may be manufactured in a few weeks, rather than 6 to 12 monthsby biological methods. The cost is far less than the cost of vaccines bycurrent biological methods.

The invention further provides preventing or treating influenza in ahuman or animal by methods comprising administering at least oneisolated or synthesized peptide of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 to the animal or human. The at least one isolated or synthesizedpeptide is administered intravenously, intramuscularly, orally,intranasally, intraocularly, via spray inhalation, or by any method ofadministration known to one of ordinary skill in the art now orhereafter. The vaccine may be administered intranasally, intraocularly,or via spray inhalation. The vaccine may be administered to a human, abird, a horse, a ferret, or a pig. The bird may be a domestic bird or awild bird and may include a chicken, a duck, a goose, or any otherdomestic or wild bird. The vaccine may be administered to a chickenincluding to a chicken at 7, 14, and 21 days after hatch.

A non-limiting vaccine of the invention is provided for, among otherthings, treatment or prevention of all strains of influenza virus. Anon-limiting vaccine of the invention may contain sequences that areconserved in strains of Low-Path H5N1, strains of High-Path H5N1, andacross other strains of influenza virus including H1N1, H1N2, H2N2,H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3, H9N2, H10N7, or any otherstrain of influenza A virus, influenza B virus, or influenza C virus.SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 of the invention have beenobserved to be conserved across many strains of influenza withparticular conservation noted in the lysine and histidine residues ofthe sequences. The lysine and histidine residues of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66 are the key amino acid residues that provide theReplikin structure of the sequences. SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66 have likewise been observed to be conserved in both High-Pathand Low-Path H5N1 and are useful in both treatment and prevention foroutbreaks of these strains of influenza as well as all other strains ofinfluenza.

Methods of Differentiating Infectivity from Lethality in Influenza Virus

One non-limiting aspect of the present invention provides methods ofdifferentiating the infectivity of an influenza virus isolate or strainof influenza virus from the lethality of the influenza virus isolate orstrain of influenza virus. Compounds for diagnostic, therapeutic, and/orpreventive purposes in influenza and therapies for the prevention andtreatment of influenza are provided based on the disclosed methods ofdifferentiation.

A method of differentiating the relative infectivity of isolate A ofinfluenza virus from the relative infectivity of isolate B of influenzavirus and the relative lethality of isolate A of influenza virus fromthe relative lethality of isolate B of influenza virus is providedcomprising: comparing the Replikin Count of the hemagglutinin proteinarea of isolate A to the Replikin Count of the hemagglutinin proteinarea of isolate B; and comparing the Replikin Count of the pB1 gene areaof isolate A to the Replikin Count of the pB1 gene area of isolate B.The relative infectivity of isolate A may be greater than, less than, orabout the same as the relative infectivity of isolate B if the ReplikinCount of the hemagglutinin protein area of isolate A is greater than,less than, or about the same as the Replikin Count of the hemagglutininprotein area of isolate B, respectively, and the relative lethality ofisolate A may be greater than, less than, or about the same as therelative lethality of isolate B if the Replikin Count of the pB1 genearea of isolate A is greater than, less than, or about the same as theReplikin Count of the pB1 gene area of isolate B, respectively.

The relative infectivity of isolate A may also be greater than, lessthan, or about the same as the relative infectivity of isolate B and therelative lethality of isolate A may not be concomitantly greater than,less than, or about the same as the relative lethality of isolate B. Therelative infectivity of isolate A may likewise be greater than therelative infectivity of isolate B and the relative lethality of isolateA may be less than or about the same as the relative lethality ofisolate B. The relative infectivity of isolate A may also be less thanthe relative infectivity of isolate B and the relative lethality ofisolate A may be greater than or about the same as the relativelethality of isolate B. The relative lethality of isolate A may also begreater than, less than, or about the same as the relative lethality ofisolate B and the relative infectivity of isolate A may be notconcomitantly greater than, less than, or about the same as the relativeinfectivity of isolate B.

A method of differentiating the relative infectivity and relativelethality of a plurality of isolates A of influenza from a given regionor time period from the relative infectivity and relative lethality ofan isolate B from a different region or different time period or fromthe relative infectivity and relative lethality of a plurality ofisolates B from a different region or different time period is alsoprovided, comprising: comparing the mean Replikin Count of thehemagglutinin protein area and the mean Replikin Count of the pB1 genearea of the plurality of isolates A to the Replikin Count of thehemagglutinin protein area of isolate B or the mean Replikin Count ofthe hemagglutinin protein area of the plurality of isolates B, and tothe Replikin Count of the pB1 gene area of isolate B or to the meanReplikin Count of the pB1 gene area of the plurality of isolates B. Aplurality of isolates A of influenza from a given region or time periodmay have a relative infectivity that is greater than, less than, orabout the same as the relative infectivity of isolate B or the pluralityof isolates B if the mean Replikin Count of the hemagglutinin proteinarea of the plurality of isolates A is greater than, less than, or aboutthe same as the Replikin Count of the hemagglutinin protein area ofisolate B or is greater than, less than, or about the same as the meanReplikin Count of the hemagglutinin protein area of the plurality ofisolates B, and the relative lethality of the plurality of isolates A isgreater than, less than, or about the same as the relative lethality ofisolate B or the plurality of isolates B if the mean Replikin Count ofthe pB1 gene area of the plurality of isolates A is greater than, lessthan, or about the same as the Replikin Count of the pB1 gene area ofisolate B or is greater than, less than, or about the same as the meanReplikin Count of the pB1 gene area of the plurality of isolates B.

The relative infectivity of the plurality of isolates A may also begreater than, less than, or about the same as the relative infectivityof isolate B or the relative infectivity of the plurality of isolates B,and the relative lethality of the plurality of isolates A may be notconcomitantly greater than, less than, or about the same as the relativelethality of isolate B or the relative lethality of the plurality ofisolates B. The relative infectivity of the plurality of isolates A mayalso be greater than the relative infectivity of the plurality ofisolates B and the relative lethality of isolate A may be less than orabout the same as the relative lethality of isolate B or the relativelethality of the plurality of isolates B. The relative infectivity ofthe plurality A of isolates may also be less than the relativeinfectivity of isolate B or the relative infectivity of the plurality ofisolates B, and the relative lethality of plurality of isolates A may begreater than or about the same as the relative lethality of isolate B orthe relative lethality of the plurality of isolates B. The relativelethality of the plurality of isolates A may also be greater than, lessthan, or about the same as the relative lethality of isolate B or therelative lethality of the plurality of isolates B and the relativeinfectivity of the plurality of isolates A may be not concomitantlygreater than, less than, or about the same as the relative infectivityof isolate B or the relative infectivity of the plurality of isolates B.

A method of differentiating the future relative infectivity of at leastone strain A of influenza virus as compared to a time T₀ from the futurerelative lethality of said at least one strain A of influenza virus ascompared to time T₀ is also provided comprising: comparing a trend ofReplikin Counts in the hemagglutinin protein area of a plurality ofisolates of strain A ending at time T₀, wherein said isolates areisolated at different time periods including time T₀, to a trend ofReplikin Counts in the pB1 gene area of a plurality of isolates ofstrain A ending at time T₀, wherein said isolates are isolated atdifferent time periods including time T₀.

The future relative infectivity of strain A may be predicted to begreater than, less than, or about the same as the relative infectivityof strain A at time T₀ if the trend of Replikin Counts in thehemagglutinin protein area of said plurality of isolates of strain A isrising, falling, or remaining about the same and the future relativelethality of strain A may be predicted to be greater than, less than, orabout the same as the relative lethality of strain A at time T₀ if thetrend of Replikin Counts in the pB1 gene area of said plurality ofisolates of strain A is rising, falling, or remaining about the same.

The future relative infectivity of strain A may also be predicted to begreater than, less than, or about the same as the relative infectivityof strain A at time T₀ and the future relative lethality of strain A maybe not concomitantly greater than, less than, or about the same as therelative lethality of strain A at time T₀. The future relativeinfectivity of strain A may also be predicted to be greater than therelative infectivity of strain A at time T₀ and the relative lethalityof strain A may be predicted to be less than or about the same as therelative lethality of strain A at time T₀. The future relativeinfectivity of strain A may also be predicted to be less than therelative infectivity of strain A at time T₀ and the relative lethalityof strain A may be predicted to be greater than or about the same as therelative lethality of strain A at time T₀.

A vaccine is also provided comprising at least one Replikin amino acidsequence from the hemagglutinin protein area of an isolate of influenzavirus and at least one Replikin amino acid sequence from the pB1 genearea of an isolate of influenza virus. The at least one Replikin aminoacid sequence from the hemagglutinin protein area may be from an isolateof influenza virus predicted to have a greater infectivity than at leastone other isolate of influenza virus and the at least one Replikin aminoacid sequence from the pB1 gene area may be from an isolate of influenzavirus predicted to have a greater lethality than at least one otherisolate of influenza virus. A vaccine may also comprise at least oneReplikin amino acid sequence from the hemagglutinin protein area and atleast one Replikin amino acid sequence from the pB1 gene area isolatedfrom (or a synthesized version of) an isolate of influenza predicted tohave a greater infectivity and a greater lethality than at least oneother isolate of influenza. A vaccine may also comprise a plurality ofReplikin peptides from the hemagglutinin protein area and a plurality ofpeptides from the pB1 gene area.

A computer readable storage medium is also provided having storedthereon instructions which, when executed, cause a processor to performa method of differentiating the relative infectivity of an influenzavirus from the relative lethality of an influenza virus. A processor mayreport the differentiation of the relative infectivity of the influenzavirus from the relative lethality of the influenza virus to a display,user, researcher, or other machine or person. The reporteddifferentiation may report whether the relative infectivity of the virusis greater than, less than, or about equal to the relative infectivityof another virus and whether the relative lethality of the virus isgreater than, less than, or about equal to the relative lethality ofanother virus.

Methods of Treating Influenza Across Strains

A method of preventing or treating influenza virus infection acrossdifferent strains is provided comprising administering at least oneisolated or synthesized protein, protein fragment, polypeptide, orpeptide comprising a homologue of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66 to an animal or human. The isolated or synthesized protein,protein fragment, polypeptide, or peptide may comprise a peptide that is30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with atleast one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. A method maycomprise administering at least one, two, three, four, five, six, seven,eight, nine, ten, eleven, or twelve or more peptide(s) that is (are)30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with atleast one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or may compriseadministering at least one, two, three, four, five, six, seven, eight,nine, ten, eleven, or twelve or more peptide(s) that consist(s) of atleast one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. A method mayfurther comprise administering at least one isolated or synthesizedprotein fragment, polypeptide, or peptide that consists of at least onepeptide A, which is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%or more homologous with at least one of the peptides of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66.

A method of preventing or treating influenza virus infection acrossstrains may also comprise administering at least one agent that iscapable of antagonizing a protein comprising a homologue of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66 wherein said agent is capable ofbinding at least a portion of said homologue that is at least 30%, 40%,50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one ofSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

Antibodies as Diagnostics and Therapies for Identified ReplikinSequences

In another aspect of the invention, isolated Replikin peptides may beused to generate antibodies, antibody fragments, or to generate oridentify other binding agents, which may be used, for example fordiagnostic purposes or to provide passive immunity in an individual.See, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 andU.S. application Ser. No. 12/010,027, filed Jan. 18, 2008 (eachincorporated herein by reference in their entirety).

Various procedures known in the art may be used for the production ofantibodies to Replikin sequences or to proteins, protein fragments,polypeptides, or peptides comprising Replikin sequences. Such antibodiesinclude, but are not limited to, polyclonal, monoclonal, chimeric,humanized, single chain, Fab fragments and fragments produced by a Fabexpression library. Antibodies that are linked to a cytotoxic agent mayalso be generated. Antibodies may also be administered in combinationwith an antiviral agent. Furthermore, combinations of antibodies todifferent Replikins may be administered as an antibody cocktail.

For the production of antibodies, various host animals or plants may beimmunized by injection with a Replikin peptide or a combination ofReplikin peptides, including, but not limited to, rabbits, mice, rats,and larger mammals. Monoclonal antibodies to Replikins may be preparedusing any technique that provides for the production of antibodymolecules. These include but are not limited to the hybridoma techniqueoriginally described by Kohler and Milstein, (Nature, 1975,256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In addition, techniques developed for the production of chimericantibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA,81:6851-6855) or other techniques may be used. Alternatively, techniquesdescribed for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce Replikin-specific single chainantibodies. Antibody fragments that contain binding sites for a Replikinmay be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)2 fragments which can be producedby pepsin digestion of the antibody molecules and the Fab fragments thatcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries can be generated(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

Binding agents are provided including an antibody, antibody fragment, orbinding agent that binds to at least a portion of an amino acid sequenceof at least one protein, protein fragment, polypeptide, or peptidecomprising at least one peptide A, where peptide A is at least 30%, 40%,50%, 60%, 70%, 80%, 90% or 95%, or 100%, homologous with at least one ofSEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. The amino acid sequence ofa protein fragment, polypeptide, or peptide may partially match theamino acid sequence of an expressed whole protein where at least one,five, ten, twenty, thirty, forty, fifty, one hundred, two hundred, threehundred, four hundred, five hundred or more amino acid residues of theamino acid sequence of the expressed whole protein are not present inthe protein fragment, polypeptide, or peptide. The amino acid sequenceof the protein fragment, polypeptide, or peptide may also partiallymatch the amino acid sequence of an expressed whole protein where atleast one, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty,ninety, one hundred, one hundred fifty, two hundred, two hundred fifty,three hundred, three hundred fifty, four hundred, four hundred fifty,five hundred, five hundred fifty or more amino acid residues of theamino acid sequence of at least one terminus of the expressed wholeprotein are not present at at least one terminus of said proteinfragment, polypeptide, or peptide.

Binding agents are also provided including an antibody, antibodyfragment, or binding agent that binds to at least a portion of an aminoacid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or morehomologous with at least one of the peptides of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66. In a non-limiting embodiment, the length ofpeptide A may be no more than one, five, ten, twenty, thirty, forty, orfifty amino acid residues longer than the sequence of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66 with which it is homologous. Bindingagents are also provided that bind to at least a portion of an aminoacid sequence of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66.

Binding agents may specifically bind to the target protein, proteinfragment, polypeptide, or peptide. Binding agents may specifically bindto a homologue of at least one of SEQ ID NO(s): 1-12, 13-20, 21-28, and32-66. Binding agents may likewise specifically bind to a peptideconsisting of any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.Binding agents may also specifically bind to a portion of a peptideconsisting of any one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66including a single amino acid within a homologue of SEQ ID NO(s): 1-12,13-20, 21-28, and 32-66, two amino acids, three amino acids, four aminoacids, five amino acids, or any number of amino acids spread within oroutside a homologue.

Nucleic Acids and Compositions of Nucleic Acids

An isolated or synthesized nucleic acid sequence is also provided thatencodes a protein, protein fragment, polypeptide, or peptide comprisingat least one peptide A, where peptide A is at least 30%, 40%, 50%, 60%,70%, 80%, 90% or 95%, or 100%, homologous with at least one of SEQ IDNO(s): 1-12, 13-20, 21-28, and 32-66. A nucleic acid sequence may alsoencode a protein, a protein fragment, a polypeptide, or a peptide wherethe amino acid sequence of the protein, protein fragment, polypeptide,or peptide partially matches the amino acid sequence of an expressedwhole protein and at least one, two, three, four, five, ten, twenty,thirty, forty, fifty, one hundred, two hundred, three hundred, fourhundred, five hundred or more amino acid residues of the amino acidsequence of the expressed whole protein are not present in the proteinfragment, polypeptide, or peptide. Further, the amino acid sequence ofthe protein, protein fragment, polypeptide, or peptide may partiallymatch the amino acid sequence of an expressed whole protein where atleast one, two, three, four, five, ten, twenty, thirty, forty, fifty,sixty, seventy, eighty, ninety, one hundred, one hundred fifty, twohundred, two hundred fifty, three hundred, three hundred fifty, fourhundred, four hundred fifty, five hundred, five hundred fifty or moreamino acid residues of the amino acid sequence of at least one terminusof the expressed whole protein may not be present at at least oneterminus of the protein, protein fragment, polypeptide, or peptide

An isolated or synthesized nucleic acid sequence may also encode apeptide consisting of 7 to about 50 amino acid residues comprising atleast one of the peptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28,and 32-66. It may also encode a peptide that is at least 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% or more homologous with at least one of thepeptide sequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. It mayalso encode a peptide consisting of at least one of the peptidesequences of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66.

The invention further provides an immunogenic composition that comprisesan isolated or synthesized nucleic acid provided above. The inventionfurther provides a vaccine against influenza comprising an isolated orsynthesized nucleic acid provided above.

Anti-Sense Nucleic Acids and siRNA

The invention further provides a nucleic acid sequence that is antisenseto a nucleic acid that encodes for any one of SEQ ID NO(s): 1-12, 13-20,21-28, and 32-66 or a small interfering nucleic acid sequence thatinterferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95% or more homologous with a nucleic acid that encodes anyone of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66 or is 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that isantisense to a nucleic acid that encodes for any one of SEQ ID NO(s):1-12, 13-20, 21-28, and 32-66.

The nucleotide sequence of the invention may be used in hybridizationassays of biopsied tissue or blood, e.g., Southern or Northern analysis,including in situ hybridization assays, to diagnose the presence of aparticular influenza strain in a tissue sample or an environmentalsample, for example. The present invention also provides kits containingantibodies specific for particular Replikins that are present in aparticular pathogen of interest, or containing nucleic acid molecules(sense or antisense) that hybridize specifically to a particularReplikin, and optionally, various buffers and/or reagents needed fordiagnosis.

Also within the scope of the invention are oligoribonucleotide sequencesthat include antisense RNA and DNA molecules and ribozymes that functionto inhibit the translation of Replikin-containing mRNA. Both antisenseRNA and DNA molecules and ribozymes may be prepared by any method knownin the art. The antisense molecules can be incorporated into a widevariety of vectors for delivery to a subject. The skilled practitionercan readily determine the best route of delivery, although generallyintravenous or intramuscular delivery is routine. The dosage amount isalso readily ascertainable.

The invention further provides antisense nucleic acid molecules that arecomplementary to a nucleic acid of the invention, wherein the antisensenucleic acid molecule is complementary to a nucleotide sequence encodinga peptide of the invention. In particular the nucleic acid sequence maybe anti-sense to a nucleic acid sequence that has been demonstrated tobe conserved over a period of six months to one or more years and/orwhich are present in a strain of influenza virus shown to have anincrease in concentration of Replikins relative to Replikinconcentration in other influenza virus strains.

The invention also provides compositions comprising RNAi-inducingentities used to inhibit or reduce influenza virus infection orreplication including small interfering RNA, which is a class of about10 to about 50 and often about 20 to about 25 nucleotide-longdouble-stranded RNA molecules. siRNA is involved in the RNA interferencepathway, where it interferes with the expression of a specific genessuch as the hemagglutinin gene or the pB1 gene area of influenza. siRNAsalso act in RNAi-related pathways, e.g., as an antiviral mechanism.

An effective amount of an RNAi-inducing entity is delivered to a cell ororganism prior to, simultaneously with, or after exposure to influenzavirus. A dosage may be sufficient to reduce or delay one or moresymptoms of influenza virus infection. Compositions of the invention maycomprise a single siRNA species targeted to a target transcript or maycomprise a plurality of different siRNA species targeting one or moretarget transcripts.

The invention provides a small interfering nucleic acid sequence that isabout 10 to about 50 nucleic acids in length and is 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% or more homologous with a nucleic acid thatencodes for any portion of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous witha nucleic acid that is antisense to a nucleic acid that encodes for anyportion of one of SEQ ID NO(s): 1-12, 13-20, 21-28, and 32-66. In afurther non-limiting embodiment, the nucleic acid sequence is about 15to about 30 nucleic acids. In a further non-limiting embodiment, thenucleic acid sequence is about 20 to about 25 nucleic acids. In afurther non-limiting embodiment, the nucleic acid sequence is about 21nucleic acids.

Advance Replikin-Based Information on Pathogenic Outbreaks Provides forRapid Production of Vaccines

Advance information concerning Replikin peptides and Replikin Peak Genesin expanding strains of pathogen allows for the rapid production ofspecific effective synthetic vaccines using one, or a combination, ofReplikin peptides or using Replikin Peak Genes. Such synthetic vaccineshave been demonstrated in rabbits, chickens, and shrimp. See, e.g.,Example 1 herein, Examples 6 and 7 of U.S. application Ser. No.11/355,120, filed Feb. 16, 2006, and Example 2 of U.S. application Ser.No. 12/108,458, filed Apr. 23, 2008. For example, a mixture of Replikinpeptides administered orally to shrimp provided up to a 91% protectiveeffect for shrimp challenged with taura syndrome virus. Taura syndromevirus is an often lethal rapidly-replicating pathogen that has asignificant negative impact on the shrimp industry.

Synthetic Replikin vaccines have also been demonstrated in the H5N1strain of influenza virus in chickens. For example, in a test ofchickens administered a mixture of twelve H5N1 Replikin peptides fromthe hemagglutinin and pB1 gene areas intranasally, intraocularly, and byspray inhalation and challenged with low pathogenic H5N1 influenzaisolated from a black duck in the state of North Carolina in the UnitedStates, a protective effect was observed at both the entry site ofinfluenza (diminished antibody production in the serum was observed ascompared to a control) and at excretion sites of influenza (influenzavirus was not observed excreted in feces or saliva from treated chickensas compared to a control). See Example 1 below.

Administration of Replikin peptides in both shrimp and chickens appearsto have provided a notable measure of mucosal immunity. For example, inExample 2 of U.S. application Ser. No. 12/108,458, a mixture of Replikinpeptides was administered by mouth to shrimp later challenged with taurasyndrome virus. The 91% protective effect of the vaccine is expected tohave been a result, at least in part, of a mucosal immune-like responsein the gut of the shrimp.

Likewise, in chickens, the administration of a mixture of Replikinpeptides provided a protective effect against entry of the H5N1 virus.For example, as may be seen in Example 1 below, three of six vaccinatedchickens, when inoculated with H5N1 virus, produced no measurable amountof antibodies against H5N1 in their serum. Instead, the virus wasapparently blocked by mucosal immunity from even entering the chickens'system. For those three chickens in which a serum immune response wasmeasured (that is, virus did enter their system), the vaccineadditionally provided a protective effect against replication of thevirus in the chickens' system (no virus was excreted in the feces orsaliva of the chickens). As such, mucosal immunity, in addition to otherimmunities, is an important aspect of the immunity imparted byReplikin-based vaccines.

Differentiation of Infectivity and Lethality in Isolates of Strains ofInfluenza Virus Provides Advance Information on Influenza Outbreaks

One aspect of the present invention provides a method of differentiatingthe infectivity of a strain of virus from the lethality of the strain ofvirus. This double differentiation of infectivity and lethality providesadvance warning of the future course of strains of influenza virus. Forexample, double differentiation of the infectivity and lethality in theH1N1 virus from 2004 through 2009 and the H5N1 virus from 2004 through2008 provides advance warning of the future course of H1N1 and H5N1 (seeFIGS. 2-5) and provides for the production of synthetic influenzaReplikin vaccines. Synthetic influenza Replikin vaccines includevaccines such as the H5N1 vaccine described in Example 1 herein.Synthetic influenza Replikin vaccines include vaccines that comprise atleast one protein, protein fragment, or peptide comprising, consistingof, homologous with, or derived from a Replikin peptide identified in ahemagglutinin protein area (the hemagglutinin protein area may beisolated from an isolate of influenza virus identified as associatedwith higher infectivity than another isolate of influenza virus) and/orat least one protein, protein fragment, or peptide comprising,consisting of, homologous with, or derived from a Replikin peptideidentified in the pB1 gene area of an isolate of influenza virus (thepB1 gene area may be associated with higher lethality than anotherisolate of influenza virus).

By isolating separate influenza virus genes in silico that differentiateinfectivity from lethality, the applicants have now provided a method ofdifferentiating the infectivity and lethality of influenza viruses. Thehemagglutinin protein area in influenza virus is now associated withinfectivity. For example, high Replikin Counts are associated withoutbreaks of various strains of influenza A virus (e.g., H1N1, H2N2,H3N2, H5N1, etc.) in the 20^(th) century.

The pB1 gene area of influenza virus is likewise now associated withlethality. For example, high Replikin Counts in the pB1 gene area areassociated with lethality in infections from H5N1 strains of influenzavirus and low Replikin Counts in the pB1 gene area are associated withlow lethality in infections from influenza. The present method fordifferentiating the infectivity and lethality of influenza viruses nowprovides both advance warning of the future course of an outbreak and abasis of production of influenza vaccines comprising synthetic Replikinpeptide sequences or comprising a protein fragment, polypeptide, orpeptide comprising Replikin peptide sequences or homologues of Replikinpeptide sequences.

The applicants have discovered that the properties of infectivity andlethality operate with a measure of independence that is differentiable.As may be seen in FIGS. 2-5 infectivity was observed to change over timein a manner different from that observed in lethality. These data applyto both H5N1 and H1N1. For example, infectivity was observed to increasein H1N1 from 2004 through 2009 while lethality was observed to remainsteady or decrease. See FIGS. 3-5. On the other hand, infectivity wasobserved to remain about the same in H5N1 from 2004 through 2008 whilelethality was observed to increase. See FIG. 2. These data reflect theepidemiological information available for each of these strains ofinfluenza over the measured years.

The data demonstrate that Replikin Counts in the hemagglutinin proteinarea of influenza shift in a manner that can be differentiated fromReplikin Counts in the pB1 gene area of influenza. The data furtherdemonstrate that infectivity and lethality are not necessarily linked ininfluenza viruses and are not necessarily linked in influenza virusesover time. Infectivity and lethality are likewise expected not to belinked between regions.

The results in FIGS. 2-5 and Examples 2-5 below provide a noteworthyverification of the validity and application of the software methodsused to determine the concentration of Replikin peptides present ingiven proteins or gene areas of influenza virus and correlate withobserved epidemiological properties in both the H1N1 and the H5N1strains of influenza virus.

The data in FIG. 5 is derived from the most recent sequence dataavailable on PubMed. A review of the data in FIG. 5 reveals that H1N1infectivity is predicted to remain high in humans in the immediatefuture and H1N1 lethality is predicted to remain low in humans for theimmediate future. The initial increase in H1N1 lethality in 2005 may berelated to an initial high lethality observed in the first cases in theoutbreak of H1N1 in or near Mexico in the spring of 2009. This increasein Replikin Count was not sustained, however, as may be seen from theReplikin Count data from 2006 through 2009. In agreement with this data,the mortality rate of subsequent cases has been observed to decline.

A review of FIGS. 2-5 likewise reveals a differentiable pattern ofchange in infectivity and lethality. As may be seen from the trend inFIG. 2, H5N1 lethality is predicted to continue increasing. Nonetheless,infectivity does not appear to increase in the immediate future. Thedata suggest that neither H1N1 in FIGS. 3-5 nor H5N1 in FIG. 2 arebecoming quiescent. The data from H1N1 in FIGS. 3-5 and for H5N1 in FIG.2 are different than data for H2N2 influenza virus and for SARScoronavirus. In H2N2 and SARS coronavirus, a decrease in Replikin Countin these infectious agents was followed by an observed quiescence intheir respective infectivity and lethality. See, e.g., U.S. applicationSer. No. 10/860,050, filed Jun. 4, 2004 (paragraph 143) and U.S.application Ser. No. 12/010,027, filed Jan. 18, 2008 (paragraph 163).

It has not previously been possible to correlate virus structures withvirus outbreaks, let alone to predict an outbreak six to twelve monthsahead of its occurrence. Such a correlation was first retrospectivelydemonstrated by the applicants monitoring Replikin Counts of wholeviruses and correlating these Replikin Counts with outbreaks andpandemics of common influenza strains that occurred over the pastcentury. The applicants then isolated in silico, a Replikin Peak Gene inthe pB1 gene area of the genome of influenza virus. The Replikin PeakGene provided advance warning of H5N1 outbreaks over the past ten years,provided advance warning of an increase in human mortality from H5N1infection, and advance warning that the increase in human mortalitywould occur in Indonesia.

Additionally, in 2008, while attention was focused on H5N1 as a possiblepandemic agent, analysis by the applicants of H1N1 sequences usingFluForecast® software (Replikins, Ltd. Boston, Mass.) warned of thecoming of an H1N1 influenza outbreak. In April of 2009, the firstreports of an outbreak of H1N1 influenza (which would eventually spreadglobally) were received from Mexico.

Presently, the higher infectivity and lower lethality of the 2009 globaloutbreak of H1N1 is tracked by relatively higher Replikin Counts in thehemagglutinin protein area of isolates of H1N1 and relatively lower anddecreasing Replikin Counts in the pB1 gene area of isolates of H1N1. SeeFIG. 5 and Table 7 below.

Likewise, the lower infectivity and higher lethality of current cases ofH5N1 globally are tracked by relatively lower Replikin Counts in thehemagglutinin protein area of isolates of H5N1 and relatively higher andincreasing Replikin Counts in the pB1 gene area of isolates of H5N1. SeeFIG. 2 and Table 3 below.

Increasing Human H1N1 Lethality Gene Replikin Count as of Jun. 8, 2009and Sep. 23, 2009

In April 2008, the applicants analyzed genomic information for isolatesof H1N1 influenza virus available at www.pubmed.com using FluForecast®software (Replikins, Ltd., Boston, Mass., USA) to determine ReplikinCounts for individual isolates in the hemagglutinin protein area and pB1gene areas. The applicants also determined the mean annual ReplikinCount for the hemagglutinin protein area and pB1 gene area. Theapplicants noted a significant increase in the Replikin Count of H1N1isolates and on Apr. 7, 2008 predicted an increased likelihood ofoutbreak and warned in a published press release that the H1N1 had nowbecome a likely candidate for the influenza strain that would cause thenext pandemic of influenza. The predicted outbreak was in fact observedin the spring of 2009. In May 2009, the applicants again analyzedgenomic information for isolates of H1N1 influenza virus and noted thatthe Replikin Count of hemagglutinin protein areas in isolates of H1N1was continuing to rise. See Example 3, Table 4, and FIG. 3. Based onthis evidence and the teachings herein, the applicants predicted thatthe 2009 outbreak would continue as a highly infective outbreak and thatH1N1 infections should be expected to be above seasonal norms in thesummer of 2009 in the Northern Hemisphere. In June 2009, the WorldHealth Organization declared the 2009 H1N1 outbreak a global pandemicand throughout the summer continued expansion of the pandemic wasreported in northern countries such as Japan, China, the U.K, and theU.S.

The applicants have now analyzed genomic information for isolates ofH1N1 influenza virus through Sep. 23, 2009. In their analysis, theapplicants have revealed that the Replikin Count of the Infectivity Genein H1N1 (white in FIG. 5) remains elevated, decreasing only 3% in itsmean since the high in April 2009. These high means provide nosignificant sign, as yet, of abatement in the current pandemic. Theapplicants have further revealed that the H1N1 Lethality Gene (black inFIG. 5), despite some activity, has not increased significantly inReplikin Count between 2001 and 2008. This absence of a significantincrease in Replikin Count in the Lethality Gene is in contrast to thelarge increases seen in the Infectivity Gene between 2001 and 2008.

Concerning the pB1 gene area, the applicants note that the StandardDeviation of the Mean (SD) (represented by capped lines in FIG. 5)increased five-fold between 2001 and December 2008. The applicantsfurther note that the Standard Deviation of the Mean increasedforty-five fold between 2001 and April 2009. This increase in standarddeviation of the mean Replikin Count indicates that some viruses in theH1N1 population are engaging in high replication rates and higherlethality.

As may be seen in FIG. 5, Replikin Count in the pB1 gene area of H1N1has gradually decreased by 38% from its high in April 2009 through toSep. 23, 2009 (p<0.001). Nevertheless, the mean Replikin Count remains15% higher and the standard deviation of the mean remains nineteen timesgreater than the level of Replikin Count seen in 2001. These higherReplikin Counts indicate that there are still individual active viruseswithin the currently circulating H1N1 virus population that containincreased Replikin Counts in their Lethality Genes. The overall trendseen in FIG. 5 since April 2009, nevertheless, is clearly toward areturn to the lower “resting” Replikin Count of about two, whichpredominated from 1980 to 2008 (or less than two, which predominatedfrom 1934 to 1979). See Table 8 below. These low Replikin Counts from1934 to 2008 were accompanied by low clinical H1N1 lethality.

Reproducibility of Replikin Counts in H1N1

Mean Replikin Counts in a wide range of viruses and organisms have beencorrelated with rapid replication and virulence. See, e.g., U.S.application Ser. No. 12/010,027, filed Jan. 18, 2008. The data in FIGS.2-5 contribute to the evidence in support of this Replikin Countphenomenon. In addition, the data in FIGS. 2-5 provide unmistakableevidence of the reproducibility of the Replikin analysis as itcorrelates with rapid replication, infectivity, and lethality in virusesand organisms. This reproducibility is particularly evident in FIG. 5where data is provided for many specific days between April andSeptember of 2009.

As described in Example 5 below, data in FIG. 5 was gathered on afrequent (sometimes daily) basis between April 2009 and September 2009.The consistent reproducibility of the Replikin Count in thehemagglutinin and pB1 gene areas over that time demonstrates measurabletrends in changes in Replikin Count such that the progression of theoutbreak and pandemic has been quantitatively observed. As may be seenin FIG. 5, these quantitative observations in concert with clinical datafrom the CDC and WHO provide further evidence of the quantitativeaccuracy of Replikin Counts in viruses such as influenza. Theobservations and clinical data also provide evidence of the ability ofthe Replikin algorithm to identify clinical changes in a pathogenicpopulation.

Synthetic Replikin Vaccine Against H1N1 and H5N2 and Other InfluenzaStrains of Virus

The differentiation of infectivity and lethality in the influenza virusgenome through the identification of concentrations of Replikinsequences in those gene areas that correlate independently withinfectivity and lethality (hemagglutinin and pB1 gene area,respectively) provides a system for attacking both the mechanics ofinfectivity and the mechanics of lethality in influenza in oneanti-virus therapy. In view of this new understanding, the applicantshave created synthetic Replikin vaccines based on homologues ofconserved Replikin sequences identified in the hemagglutinin proteinarea and homologues of conserved Replikin sequence identified in the pB1gene area of influenza.

One vaccine was initially engineered from sequences identified in theLow-Path H5N1 isolated from the black duck in North Carolina, USA andconfirmed to be conserved in both Low-Path and High-Path H5N1 strains aswell as across influenza strains with conservation particularly noted inthe key amino acid residues of the Replikin sequence, namely, lysine andhistidine amino acid residues. The vaccine was designed to deliver anapproximately equal-parts-by-weight mixture of twelve Replikin peptidesto the immune system of an animal or human. Six of the peptides wereisolated in silico from the hemagglutinin protein area and six of thepeptides were isolated in silico from the pB1 gene area. All twelvepeptides were then synthesized, and combined in a vaccine.

As described in Example 1 below, a vaccine has now successfullyprotected chickens from low-pathogenic H5N1 infection and hassuccessfully blocked excretion of low-pathogenic H5N1 virus frominfected chickens. The vaccine was developed from influenza Replikinpeptides shared between influenza strains and conserved for decadeswithin influenza strains and was engineered as a mixture of twelveReplikin peptides identified as expressed from the genome of H5N1 virus.Six of the Replikin peptides were synthesized according to sequencesisolated from the hemagglutinin protein area of H5N1, which is involvedin attachment and entry of influenza virus into a cell. Six of theReplikin peptides were synthesized according to sequences isolated fromthe pB1 gene area of H5N1, which has been identified as involved inreplication of influenza virus in a host cell.

Another exemplary vaccine has been designed from sequences identified asconserved in H1N1 isolates. The sequences are likewise conserved acrossstrains with conservation particularly noted in the key amino acidresidues of the Replikin sequence, namely, lysine and histidine aminoacid residues. The vaccine was designed to deliver an approximatelyequal-parts-by-weight mixture of eight Replikin peptides to the immunesystem of an animal or human. All eight peptides are synthesized, andcombined in a vaccine.

The peptide mixture may be administered in any manner known to one ofskill in the art including with a pharmaceutically acceptable carrier.Administration may be intraocularly, intranasally, transdermally,intramuscularly, or via any method of administration known now orhereafter to one of skill in the art. Because the vaccine is based oninfluenza Replikin peptides shared between influenza strains andconserved for decades within influenza strains, the vaccine may beadministered as a therapy against infection by any influenza virusinfection harboring conserved Replikin peptides sharing homology with atleast one peptide of the vaccine. Such strains include any strain ofinfluenza A, B, or C. The vaccine may be administered, for example,against H1N1, H1N2, H2N2, H3N2, H3N8, H5N1, H5N2, H7N7, H7N2, H7N3,H9N2, or H10N7 strains of influenza virus. The vaccine may be furtheradministered against H5N1, H5N2, H3N2, H9N2, or H1N1.

Therapies Against Possible Combination of H1N1 and H5N1

In both H1N1 and H5N1, the applicants have observed that Replikinsequences are distributed unevenly throughout the genome. Instead of aneven distribution, Replikin sequences are concentrated in two regions ofthe genome, the hemagglutinin protein area and the pB1 gene area. Thesegene areas are associated with infectivity and lethality, respectively,in H1N1 as well as H5N1 and other influenza strains. Clinically, H1N1 isknown to have high infectivity and low lethality in 2009 while H5N1 isknown to have low infectivity and high lethality (for example, H5N1lethality in humans has reached 80% in recent outbreaks).

FIGS. 2-5 provide examples of the association of Replikin Count inhemagglutinin with infectivity and Replikin Count in the pB1 gene areawith lethality. The principle that Replikin Count in a genome could beassociated with lethality has been quantitatively measured in apredictive study of the relative lethality of four strains of TauraSyndrome virus. See U.S. application Ser. No. 12/010,027, filed Jan. 18,2008. The applicability of this principle to anti-viral therapies wasdemonstrated when Taura Syndrome virus was blocked by a specificsynthetic Replikin sequence vaccine, protecting 91% of challenged shrimpfrom pathogenic mortality. In another example, an increase in thelethality of H5N1 in human cases in 2007 and 2008 was predicted inadvance by the strain-specific Replikin Count of the Lethality Gene ofH5N1. See id. Similarly, by comparing the Replikin Counts of the pB1gene area of H5N1 Genes in eight Asian countries in 2006, the geographicsite which would be first and worst struck in 2007 was correctlypredicted as Indonesia. See id.

Because of the known ability of segments of the genomic sequences totransfer between influenza strains, public health officials areconcerned about a possibility that the high infectivity of H1N1 might becombined with the high lethality of H5N1. At present, there isapparently no method available to predict the probability of thisoccurrence. Nevertheless, to prepare in advance for the possibility ofan H1N1-H5N1 combination, the applicants have developed a syntheticReplikin sequence vaccine based on Replikin structures shared in thecommon Influenza A strains.

Example 1 Synthetic Replikin Vaccines Block H5N1 in Chickens

A synthetic Replikin vaccine containing an approximatelyequal-parts-by-weight mixture of twelve H5N1 Replikin peptides wastested in chickens against a low pathogenic strain of H5N1 isolated froma black duck in North Carolina, USA. Low-Path H5N1 strains infectmigratory birds and impair health and productivity of commercial flocksof U.S. chickens, usually with little mortality in the commercialflocks. These Low-Path H5N1 strains are very closely related in virusstructure to their more lethal High-Path H5N1 relatives in Eurasia. Amutation from a Low-Path to a High-Path strain has so far not beenobserved but mutations of this type over time may be expected by one ofskill in the art.

The tested vaccine was engineered from sequences identified in theLow-Path H5N1 and confirmed to be conserved in both Low-Path andHigh-Path H5N1 strains over decades as well as across influenza strainswith conservation particularly noted in the key amino acid residues ofthe Replikin sequence, namely, lysine and histidine amino acid residues.The tested vaccine was engineered to block both the entry site of H5N1virus and the replication site of those H5N1 viruses that manage toenter into host cells. As such, the vaccine is called the TWO-PUNCHvaccine. As demonstrated below, evidence from the described test of theTWO-PUNCH vaccine in chickens suggests that both mechanisms for whichthe vaccine was designed were effective: (1) virus entry into inoculatedchickens was diminished by immunity from the vaccine and (2) virusreplication within infected cells was sufficiently limited by immunityfrom the vaccine to block excretion of the virus in feces of testedbirds.

The vaccine comprises a mixture of twelve Replikin peptides. Six of theReplikin peptides are synthesized according to sequences isolated fromthe hemagglutinin protein area of H5N1, which is involved in attachmentand entry of influenza virus into a cell. Six of the Replikin peptidesare synthesized according to sequences isolated from the pB1 gene areaof H5N1, which has been identified as involved in replication ofinfluenza virus in a host cell.

The following six Replikin sequences contained in the vaccine wereisolated from the hemagglutinin protein area:

(SEQ ID NO: 1) (1) HAQDILEKEHNGKLCSLKGVRPLILK; (SEQ ID NO: 2) (2)KEHNGKLCSLKGVRPLILK; (SEQ ID NO: 3) (3) KKNNAYPTIKRTYNNTNVEDLLIIWGIHH;(SEQ ID NO: 4) (4) HHSNEQGSGYAADKESTQKAIDGITNK; (SEQ ID NO: 5) (5)HDSNVKNLYDKVRLQLRDNAK; and (SEQ ID NO: 6) (6) KVRLQLRDNAKELGNGCFEFYH.

The following six Replikin sequences contained in the vaccine wereisolated from the pB1 gene area:

(SEQ ID NO: 7) (1) KDVMESMDKEEMEITTH; (SEQ ID NO: 8) (2)HFQRKRRVRDNMTKK; (SEQ ID NO: 9) (3) KKWSHKRTIGKKKQRLNK; (SEQ ID NO: 10)(4) HKRTIGKKKQRLNK; (SEQ ID NO: 11) (5) HEGIQAGVDRFYRTCKLVGINMSKKK; and(SEQ ID NO: 12) (6) HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK.

The vaccine comprises an approximate equal-parts-by-weight mixture ofthe twelve peptides. The following peptide amounts were combined tocreate an initial mixture of the vaccine:

(SEQ ID NO: 1) HAQDILEKEHNGKLCSLKGVRPLILK 239.6 mg (SEQ ID NO: 2)KEHNGKLCSLKGVRPLILK 200.8 mg (SEQ ID NO: 3)KKNNAYPTIKRTYNNTNVEDLLIIWGIHH 213.0 mg (SEQ ID NO: 4)HHSNEQGSGYAADKESTQKAIDGITNK 135.6 mg (SEQ ID NO: 5)HDSNVKNLYDKVRLQLRDNAK 170.8 mg (SEQ ID NO: 6) KVRLQLRDNAKELGNGCFEFYH188.3 mg (SEQ ID NO: 7) KDVMESMDKEEMEITTH 161.9 mg (SEQ ID NO: 8)HFQRKRRVRDNMTKK 138.3 mg (SEQ ID NO: 9) KKWSHKRTIGKKKQRLNK 217.8 mg(SEQ ID NO: 10) HKRTIGKKKQRLNK 178.0 mg (SEQ ID NO: 11)HEGIQAGVDRFYRTCKLVGINMSKKK 159.2 mg (SEQ ID NO: 12)HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK 233.8 mgThe total amount of the mixture was 2237.1 mg.

The peptide mixture was then divided into three equal parts foradministration of the vaccine on three different days following hatch(days 1, 7, and 28). After dissolution with water, the three equal partswere administered to individual birds in two groups of 20 birds each fora total administration on each day of 40 birds. The total amount ofactive peptide ingredient administered to each bird at the time ofadministration (either intranasally and intraocularly or via sprayinhalation) was about 18.6 mg per bird per administration. The vaccinesolution was administered to chickens intranasally at a firstadministration on day 1 after hatch, intraocularly at a secondadministration on day 7 after hatch, and via fine spray inhalation at athird administration on day 14 after hatch.

Chickens on the first day of life were separated into four groups withtwenty chickens per group. The first group was a control group notvaccinated and not challenged with Low-Path H5N1. The second group wasvaccinated and not challenged with Low-Path H5N1. The third group wasvaccinated and subsequently challenged with Low-Path H5N1 on day 28after hatch. The fourth group was not vaccinated and was challenged withLow-Path H5N1 on day 28 after hatch.

Vaccinated chickens were subject to the vaccine on days 1, 7, and 21after hatch as described above. Challenged chickens were inoculated withLow-Path H5N1 virus in the soft palate on day 28 after hatch. Serum fromselected chickens was analyzed in all groups for antibodies against theH5N1 virus on days 7, 14, and 21 following challenge (days 35, 42, and49 after hatch). PCR for virus fecal excretion was also analyzed for allgroups.

Unvaccinated control chickens demonstrated both an expected high virusentry (as indicated by a high titer of antibodies against H5N1) and anexpected high virus replication (as indicated by high fecal and salivalexcretion of the virus detected by PCR). In contrast, the vaccinatedchickens demonstrated lower virus entry (as indicated by a low titer ofantibodies against H5N1 or by the observation of no antibodies againstH5N1 in serum) and an absence of fecal or salival excretion of virusindicating low or no virus replication in the vaccinated chickens. Thedata suggest, therefore, that the virus was partially blocked on entryby the chickens' immune response to the vaccine and the limited amountof virus that did enter the chickens' system was blocked from sufficientreplication in the chickens' host cells to excrete virus in the feces orsaliva.

The data in Table 1 below provide the numbers of chickens tested in eachof the four groups (Negative Control, Vaccinated, Vaccinated andChallenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (notvaccinated)) on a particular test day and the numbers of chickens inwhich production of antibodies to H5N1 was detected with a serum titer.

TABLE 1 Serum Antibody Test of Low-Path H5N1 Challenge of VaccinatedChickens Day 7 Day 14 Day 21 (Chickens (Chickens (Chickens ProducingProducing Producing Antibody Antibody Antibody GROUP to H5N1) to H5N1)to H5N1) Negative Control 0 of 7 0 of 7 0 of 7 Vaccinated 0 of 7 6 of 60 of 5 Vaccinated and 1 of 7 3 of 6 2 of 7 Challenged with Low- PathH5N1 Challenged with Low- 4 of 7 7 of 9 3 of 9 Path H5N1

The data in Table 2 below provide the number of chickens tested for H5N1virus in their saliva and feces in each of the four groups (NegativeControl, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, andChallenged with Low-Path H5N1 (not vaccinated)) on a particular test dayand the numbers of chickens in which H5N1 was detected in their fecesand saliva based on PCR analysis.

TABLE 2 PCR Test for Excreted H5N1 Virus from Low-Path H5N1 Challenge ofChickens Day 7 Day 14 Day 21 (Chickens (Chickens (Chickens ProducingProducing Producing Antibody Antibody Antibody GROUP to H5N1) to H5N1)to H5N1) Negative Control  0 of 10 0 of 7 0 of 7 Vaccinated  0 of 10 0of 7 0 of 7 Vaccinated and 0 of 7 0 of 7 0 of 7 Challenged with Low-Path H5N1 Challenged with Low- 3 of 7 2 of 9 1 of 7 Path H5N1

The data in Tables 1 and 2, demonstrate the effectiveness of thedouble-protective mechanism of the TWO-PUNCH vaccine. First, whileseveral non-vaccinated chickens challenged with H5N1 excreted virus intheir feces and saliva, no vaccinated chickens challenged with H5N1excreted virus in their feces or saliva. See Table 2. These datademonstrate that the vaccine provided a protective effect againstreplication of the virus. Second, while four of seven unvaccinatedchickens challenged with H5N1 were producing serum antibodies againstH5N1 on day 7, seven of nine unvaccinated chickens challenged with H5N1were producing serum antibodies against H5N 1 on day 14, and three ofnine unvaccinated chickens challenged with H5N1 were producing serumantibodies against H5N1 on day 28, only one of seven vaccinated andchallenged chickens was producing serum antibodies against H5N1 on day7, only three of six vaccinated and challenged chickens were producingserum antibodies against H5N1 on day 14, and only two of sevenvaccinated and challenged chickens were producing serum antibodiesagainst H5N1 on day 21. See Table 1. These data demonstrate that forsome of the vaccinated chickens, the H5N1 virus challenge was stoppedprior to entry into the chicken's system (likely by antibodies producedat the mucus membranes). These data further demonstrate that for thosevaccinated and challenged chickens in which the virus entered the system(resulting in production of serum antibodies), the virus was nonethelessnot excreted in feces or saliva.

As may be seen from the data in Table 1, almost all of thenon-vaccinated challenged birds seroconverted (producing detectableantibody). This demonstrates infection of the non-vaccinated birds. Onthe other hand, only some of the vaccinated challenged birdsseroconverted. Further, for those vaccinated birds that did seroconvert,the antibody titers were low. Additionally, the negative control grouphad no seroconversion. These data demonstrate a protective effect of thevaccine on the birds. Additionally, Table 2 demonstrates the absence ofdetectable influenza in the feces and saliva of vaccinated birds. Thatviral excretion was blocked by this influenza Replikins vaccine isparticularly significant because it is generally acknowledged that themaintenance of reservoirs of H5N1 virus in flocks of migratory birds anddomestic chickens in both Asia and the U.S. (and the regional spread ofH5N1 virus from these reservoirs) is dependent on viral excretionspicked up by neighboring chickens and birds. Regardless of the level oflethality of a strain of H5N1 virus, absent excretion of virus, there isexpected to be no spread of the virus. As such, data observed fromadministration of the TWO-PUNCH Replikin peptide vaccine in chickensdemonstrates the efficacy of the vaccine as (1) a barrier to entry ofthe virus, (2) a block of replication of the virus, and (3) a block offecal spread of the virus.

In a recent peer-reviewed publication by Jackwood et al. concerning thevaccine (Avian Diseases,” Publication Online:http://avdi.allenpress.com/avdionline/?request=get-abstract&doi=10.1637%2F8892-042509-ResNote.1;Hard copy Article in Press. Jul. 4, 2009), the authors conclude: “Takentogether, these data indicate that a Replikin peptide vaccinespecifically made against the H5N1 Black Duck/NC/674-964/06 andadministered three times to the upper-respiratory tract, was capable ofprotecting chickens from infection and shedding of the homologous virus,which is extremely important because reduced virus shedding andtransmission decreases the potential for H5 LPAI viruses to become HPAIviruses. The study is also important because it shows that the vaccinecan be effectively mass delivered to the upper-respiratory tract.” Id.

Because of shared sequences, the vaccine may likewise be administeredagainst H5N2, H1N1, H9N2, H3N2 or any other influenza strain havingReplikin sequences that share homology with the peptides of the vaccine.

Example 2 Differentiation of Infectivity and Lethality in H5N1 Isolatesfrom 2004 Through 2008

The infectivity and lethality of isolates of the H5N1 influenza virusfrom between 2004 and 2008 was differentiated by analyzing the ReplikinCounts of sequences of the hemagglutinin protein area of isolatespublicly available at www.pubmed.com for the years 2004 to 2008 and theReplikin Counts of sequences of the pB1 gene area of isolates publiclyavailable at www.pubmed.com for the years 2004 to 2008.

The Replikin Count (number of Replikin sequences per 100 amino acidresidues of a sequence) of the publicly available hemagglutininsequences and the publicly available pB1 gene area sequences wereanalyzed using FLUFORECAST® software (Replikins, Ltd., Boston, Mass.).The results of the analysis are provided below in Table 3.

TABLE 3 H5N1 Influenza Virus Infectivity and Lethality pB1 Gene AreaMean Hemagglutinin Annual Mean Annual Hemagglutinin Replikin pB1 GeneArea Replikin Count Standard Sequences Count Standard Sequences Year(Infectivity) Deviation Analyzed (Lethality) Deviation Analyzed 2004 4.20.6 17 2 0.1 14 2005 3.8 0.2 14 1.8 0.1 6 2006 3.8 0.4 29 5.9 7.0 242007 4.6 0.5 27 12.2 7.9 33 2008 4.5 0.4 6 15.1 6.5 6

As may be seen from the data in Table 3 and the illustration of the datain FIG. 2, analysis of the Replikin Counts of hemagglutinin protein areasequences and pB1 gene area sequences for isolates of H5N1 from 2004through 2008 reveal that Replikin Counts in the hemagglutinin proteinarea and Replikin Counts in the pB1 gene area are independent from oneanother in isolates from a given year and Replikin Counts in these areasof the genome trend in directions that are independent from one another.As may be observed, mean annual Replikin Counts in the pB1 gene areatrended upward while mean annual Replikin Counts in the hemagglutininprotein area remained about the same.

Replikin Counts in the pB1 gene area are associated with lethality andReplikin Counts in the hemagglutinin protein area are associated withinfectivity. As such, the data in Table 3 as illustrated in FIG. 2demonstrate that lethality was increasing from 2004 through 2008 whileinfectivity was fairly steady. The data correlate with epidemiologicaldata in H5N1. For example, H5N1 has continued to cause high rates ofmortality in humans. The highest presently recorded lethality waspredicted by the applicants following analysis of publicly availableH5N1 pB1 sequences from isolates in 2005 and 2006. In 2006 theapplicants predicted that mortality rates would increase in H5N1infections in response to increasing Replikin Counts in the pB1 genearea of the virus. The Applicants further predicted that increasedmortality rates would particularly affect Indonesia because ReplikinCounts were notably rising in that country. As predicted, H5N1 viralinfection resulted in the death of as many as 80% of infected hosts inIndonesia in 2007.

These high rates of lethality have not greatly diminished globally. Infact, the World Health Organization estimates the mortality rate of thepresent H5N1 outbreak at around 60%. The lethality of the virus, assuch, remains high and epidemiological data agrees with the ReplikinCount data illustrated in FIG. 2 in that neither set of data suggeststhat lethality will decrease in the near future.

The infectivity of H5N1 influenza virus has apparently remained steadyover the years from 2004 through 2008 with very low rates of infectionand very low rates of possible transmission between humans. Inparticular, because of infrequent infections in humans, the H5N1 virusproduced less than 300 World Health Organization confirmed deaths overthe 10 years through the spring of 2008 even though the virus killed asmany as 60% of those infected. For H5N1, the high human mortality rate,in combination with a low infectivity, appear to limit the ability ofH5N1 to presently produce an influenza pandemic.

Nevertheless, the data illustrated in FIG. 2 predict that H5N1 is notentering a quiescent phase but will continue with high lethality and lowinfectivity in the near future. This prediction of continued lethalityis in contrast to previous predictions of quiescence in the H2N2 strainof influenza virus and in SARS. See U.S. application Ser. No.10/860,050, filed Jun. 4, 2004 (paragraph 143) and U.S. application Ser.No. 12/010,027, filed Jan. 18, 2008 (paragraph 163).

Example 3 Differentiation of Infectivity and Lethality at Outset of 2009Global Outbreak of H1N1 Influenza Virus (Spring 2009)

The infectivity and lethality of the H1N1 influenza virus causing the2009 global outbreak of H1N1 influenza virus was differentiated byanalyzing the Replikin Counts of sequences of the hemagglutinin proteinarea of isolates of H1N1 publicly available at www.pubmed.com for theyears 2004 through the spring of 2009 and the Replikin Counts ofsequences of the pB1 gene area publicly available at www.pubmed.com forthe years 2004 through the spring of 2009.

The Replikin Count (number of Replikin sequences per 100 amino acidresidues of a sequence) of the publicly available hemagglutininsequences and the publicly available pB1 gene area sequence wereanalyzed using FLUFORECAST® software (Replikins, Ltd., Boston, Mass.).The results of the analysis are provided below in Table 4.

TABLE 4 H1N1 Influenza Virus Infectivity and Lethality pB1 Gene AreaMean Hemagglutinin Annual Mean Annual Hemagglutinin Replikin pB1 GeneArea Replikin Count Standard Sequences Count Standard Sequences Year(Infectivity) Deviation Analyzed (Lethality) Deviation Analyzed 2004 5.31.4 51 2 0.0 1 2005 6.2 2.1 160 4.6 6.5 6 2006 6.2 1.9 234 2.2 0.4 32007 6.2 1.5 680 2.1 0.1 15 2008 7 0.9 491 2 0.0 25 2009 10 2.4 29 2 0.01

As may be seen from the data in Table 4 and the illustration of the datain FIG. 3, analysis of the Replikin Counts of hemagglutinin protein areasequences and the pB1 gene area sequences for isolates of H1N1 from 2004through 2009 reveal Replikin Counts in the hemagglutinin protein areaand Replikin Counts in the pB1 gene area are independent from oneanother in isolates from a given year and Replikin Counts in these areasof the genome trend in directions that are independent from one another.Mean Replikin Counts in the hemagglutinin protein area trended upwardwhile mean annual Replikin Counts in the pB1 gene area stayed about thesame with a noticeable spike in mean annual Replikin Count in the pB1gene area in 2005.

Replikin Counts in the hemagglutinin protein area are associated withinfectivity and Replikin Counts in the pB1 gene area are associated withlethality. As such, the data illustrated in FIG. 3 demonstrate thatinfectivity was on the increase from 2004 through 2009 while lethalitywas fairly steady with a noticeable increase around 2005. The datacorrelate with epidemiological data from the global 2009 outbreak ofH1N1. For example, the global outbreak of the H1N1 strain of influenzavirus in the spring of 2009 has been observed to have high infectivitywith effective transmission from host to host and the potential for anefficient and rapid spread of the virus internationally. Seewww.cdc.gov/mmwr/preview/mmwrhtml/mm5817a1.htm. Despite rapid globaltransmission of the virus, the case-fatality rate remains low andinfection by the virus generally has been observed to be mild,self-limited, and uncomplicated. Id. Nevertheless, because the H1N1virus has not been wide-spread in the population over the past years,some cases of severe disease and death have been reported in previouslyhealthy young adults and children. Id.

By monitoring the Replikin Count in the whole genome of H1N1 in thespring of 2008, the inventors predicted the outbreak of H1N1 in thespring of 2009, which has now become the global outbreak of 2009. Inparticular, a review of publicly available sequences from isolates ofthe H1N1 strain of influenza virus in the spring of 2008 revealed anincrease in mean Replikin Count (Replikin sequences per 100 amino acidsin the publicly available sequence) in the hemagglutinin protein area ofisolates of H1N1 to a mean of 7.6 with a standard deviation ofplus/minus 1.4. The mean Replikin Count of 7.6 represented the highestReplikin Count in H1N1 influenza virus since the 1918 H1N1 pandemic. Thep value for the observation that the Replikin Count was the highestsince the 1918 H1N1 pandemic was less than 0.001. The applicants notedthat the increase in Replikin Count in isolates of H1N1 appeared to bespecific to H1N1 in that a concurrent 80% decline in the Replikin Countof H3N2 was observed.

The applicants noted in concert with the historically high ReplikinCount that H1N1 influenza virus appeared to be rapidly replicatingsimultaneously in the U.S. and Austria. Based on theobserved-historically-high Replikin Counts, the applicants predictedthat H1N1 should succeed H5N1 as the leading candidate for the nextexpected and overdue pandemic. The applicants noted, however, thatcertain virus Replikin structures detected in all three previouspandemics, namely, 1918 H1N1, 1957 H2N2, and 1968 H3N2, as well as inH5N1, had not yet been detected in the evolving H1N1 isolates in thespring of 2008. The applicants noted that the 1918 H1N1 outbreak had anestimated human mortality rate of about 2.5 to 10%. Despite thismoderate mortality rate, a very high infectivity rate in the 1918pandemic produced an estimated 50 million deaths worldwide.

The lethality of H1N1 influenza virus has apparently remained generallysteady over the years from 2004 through 2009. There appears to have beena spike in lethality in Mexico, however, just at the beginning of thespring 2009 outbreak. This spike in lethality appears to have waned asthe outbreak spread in Mexico and globally. This initial spike may berelated to H1N1 isolates carrying a high Replikin Count in the pB1 genearea as reflected in the 2005 isolates disclosed in Table 4 above.However, as the outbreak spread, the 2005 increase in Replikin Count inthe pB1 gene area was apparently lost and the mortality rate ofsubsequent cases also declined.

The data in Table 4, additionally predict that H1N1 is not entering aquiescent phase but will continue with high infectivity in the nearfuture.

Example 4 Double Differentiation of Infectivity and Lethality in 2009Global Outbreak of H1N1 Influenza Virus Through Jun. 8, 2009

The infectivity and lethality of the H1N1 influenza virus causing the2009 global outbreak of H1N1 influenza virus was differentiated byanalyzing the Replikin Counts of sequences of the hemagglutinin proteinarea of isolates of H1N1 publicly available at www.pubmed.com from 2001through Jun. 8, 2009 and the Replikin Counts of sequences of the pB1gene area publicly available at www.pubmed.com from 2001 through Jun. 8,2009.

The Replikin Count (number of Replikin sequences per 100 amino acidresidues of a sequence) of the publicly available hemagglutininsequences and the publicly available pB1 gene area sequences wereanalyzed using FLUFORECAST® software (Replikins, Ltd., Boston, Mass.).The results of the analysis are provided below in Table 5.

TABLE 5 H1N1 Influenza Virus Infectivity and Lethality in Humans pB1Gene Area Mean Hemagglutinin Annual Mean Annual Hemagglutinin ReplikinpB1 Gene Area Replikin Count Standard Sequences Count Standard SequencesYear (Infectivity) Deviation Analyzed (Lethality) Deviation Analyzed2001 4.3 1.9 144 2 0.1 122 2002 3.5 1.9 62 2 0.1 4 2003 4.8 1.3 88 2 0.225 2004 3.1 3 15 2 0.1 6 2005 5.1 2.9 68 2.6 3.7 19 2006 5.6 1.5 102 2.10.7 27 2007 6 1.6 537 2.1 1.1 318 2008 6.7 1.3 320 2 0.2 41 2009 9.7 1.9357 3.2 3.7 177

As may be seen from the data in Table 5 and the illustration of the datain FIG. 4, analysis of the Replikin Counts of hemagglutinin protein areasequences and the pB1 gene area sequences for isolates of H1N1 from 2001through Jun. 8, 2009 reveal Replikin Counts in the hemagglutinin proteinarea and Replikin Counts in the pB1 gene area are independent from oneanother in isolates from a given year and Replikin Counts in these areasof the genome trend in directions that are independent from one another.As may further be seen in Table 5 and FIG. 4, mean annual ReplikinCounts in the hemagglutinin protein area trended upward from 2001through 2009 while mean annual Replikin Counts in the pB1 gene areastayed about the same with a small spike in mean annual Replikin Countin 2005 that was not statistically significant (p<0.40) and a notablespike in mean annual Replikin Count in 2009 that is statisticallysignificant (p<0.001).

The data in Table 5 demonstrate that infectivity was on the increasefrom 2001 through 2009 while lethality was fairly steady through 2008.The same pattern of steady Replikin Counts related to lethality is alsoseen in the 2004 through May 18, 2009 data provided in Example 3 above.

An increase is additionally observed in the data from 2009 in Table 5,which demonstrate a notable increase in mean annual Replikin Count forthe pB1 gene area from 2 (+/−0.2) in 2008 to 3.2 (+/−3.7) in 2009. Inanalyzing 836 isolates of H1N1 influenza virus isolated over the past 76years, the applicants have observed that the Replikin Count of the pB1gene area has generally been in the range of about two Replikinsequences per 100 amino acids for around 76 years. See Table 6. The 2008through 2009 increase from 2 to 3.2 (with a large increase in standarddeviation) represents, therefore, a notable change in the lethality ofthe H1N1 influenza virus.

The infectivity and lethality data for 2009 differs in Table 5 abovefrom the data in Table 4 above in that the data in Table 5 represent themost recent genomic sequences published at www.pubmed.com as of Jun. 8,2009. The data in Table 4 above represent a much smaller number ofgenomic sequences published at www.pubmed.com only through May 18, 2009.

While the data in Table 5 demonstrate an increase in mean annualReplikin Count for the pB1 gene area from 2 (+/−0.2) in 2008 to 3.2(+/−3.7) in 2009, the data in Table 4 demonstrate a steady mean annualReplikin Count for the pB1 gene area from 2 (+/−0) in 2008 to 2 (+/−0)in 2009. As such, the increase in Replikin Count in Table 5 above, ascompared to Table 4, reflects an increase in mean Replikin Count forisolates published at www.pubmed.com between May 18, 2009 and Jun. 8,2009. This increase in Replikin Count for genomic information publishedover a three week period demonstrates a rise in lethality in theevolving virus. The data in Table 5, predicted that H1N1 was notentering a quiescent phase but would continue with high infectivity andpossible increasing lethality in the future.

The following accession numbers disclosed in Table 6 were queried by theapplicants at www.pubmed.com using FLUFORECAST® software (Replikins,Ltd., Boston, Mass.) through Jun. 8, 2009. Mean annual Replikin Count,standard deviation, and statistical p-values for each year are reported.The Replikin Counts from these accession numbers are generally reflectedin the data in Table 5 and FIG. 4.

TABLE 6 H1N1 Annual Mean Replikin Count Mean No. of Replikin IsolatesCount Year PubMed Accession Number-Replikin Count per year per year S.D.Significance 1933 ABD77804; ACF54606; ABF47963 3 2.4 0.0 low p < .0011934 ACF41842; ABD77683 2 1.6 0.0 low p < .001, prev p < .001 1935ABD62789; ABO38392; ABN59420 3 1.6 0.2 low p < .20, prev p > .50 1936ABO38359 1 1.5 0.0 prev p < .20 1940 ABI20834 1 1.5 0.0 1942 ABD62850 11.2 0.0 1943 ABD79109; ABO38381; ABO38062 3 1.5 0.0 low p > .50, prev p< .001 1945 ABP49335 1 1.5 0.0 prev p > .50 1946 ABD79120 1 1.5 0.0 1947ABD77815 1 1.5 0.0 1948 ABN59409 1 1.6 0.0 1949 ABN59442 1 1.6 0.0 1950ABD61743; ABP49324 2 1.6 0.0 low p < .001 1951 ABR15816; ABQ44479;ABQ01319; ABP49489 4 1.6 0.0 low p < .001 1954 ABD60974; ABO52288 2 1.50.0 prev p < .001 1957 ABD15267 1 1.6 0.0 prev p < .001 1976 ACQ99829;ABV45846; ABQ44402 3 1.9 0.2 low p < .02, prev p < .05 1977 ABD95358;ABD60952; ABD60941; ABO44142 4 1.5 0.1 low p < .30, prev p < .002 1978ABY81357; ABP49456; ABP49346; ABO38073; ABO33000; ABO32989; 14 1.4 0.3low p < .40, ABN59431; ABK79956; ABG26821; ABF47745; ABF4; prev p < .207734; ABF47723; ABF47712; ABF47701 1979 ABW36319; ABQ01330; ABN50764 31.8 0.0 low p < .001, prev p < .001 1980 ABO38370; ABO33017; ABF47756; 32.0 0.0 low p < .001, prev p < .001 1981 ABO52266 1 2.0 0.0 prev p > .501982 ABD95347; ABD77826; ABO52805 3 2.2 0.2 low p < .02, prev p < .101983 ABW91193; ABO38348; ABO37996; ABO33033; ABN50925; ABN50908; 47 2.00.1 low p < .001, ABM66894; ABM66916; ABM66905; ABM22243; ABM22232; prevp < .05 ABM22221; ABM22210; ABM22199; ABM22188; ABM22177; ABM22166;ABL67272; ABL67261; ABK80055; ABK80044; ABK80033; ABK40609; ABK40598;ABK40587; ABK40576; ABK40565; ABK40554; ABK40543; ABK40518; ABI92310;ABI30386; ABI20867; ABG88352; ABG88341; ABF47833; ABF47778; ABG79960;ABF47855; ABF47844; ABF47767; ABF47800; ABG26843; ABG26832; ABF47822;ABF47811; ABF47789 1984 ABP49357; ABO38414 2 2.0 0.0 low p < .001, prevp < .04 1986 P03430; P03431; ABP49368; ABO44131; ABO38403; ABM22254; 72.0 0.3 low p < .001, P03427 prev p > .50 1987 ABQ44424; ABN50948;ABN50936 3 2.0 0.0 low p < .001, prev p > .50 1988 ABU80408 1 2.1 0.0prev p < .001 1989 ACL12269; ACK99451 2 2.0 0.0 low p < .001, prev p <.001 1990 P16512; P16510; P16514; P18882; P16502 5 2.1 0.4 low p < .02,prev p > .50 1991 ABD60963; ACQ84485; ACF41941; ACF41930 4 1.9 0.2 low p< .02, prev p < .30 1993 NP_040985; AAA43643; AAA43641; AAA43640;AAA43639; AAA43582; 7 2.1 0.4 low p < .002, AAA43581 prev p < .20 1995ACK99473; ACF41875; ABG88330; ABG26799; ABF47646; ABJ53446; 24 2.0 0.1low p < .001, ABI92321; ABI30375; ABI20878; ABI20845; ABG88319; prev p <.30 ABG88308; ABF47635; ABG47848; ABG26788; ABF47613; ABE26999;ABE12040; ABE11968; ABE11930; ABE11908; ABE11897; ABE11886; ABE118751996 ABO52233; ABO38018; ABN51074; ABN50981; ABN50970; ABN50959; 27 2.00.1 low p < .001, ABF47657; ABM22298; ABM22287; ABM22276; prev p > .50ABM22265; ABJ53512; ABJ53501; ABI95291; ABI95280; ABI95269; ABI95258;ABI93036; ABI21582; ABI21571; ABI21560; ABI21549; ABI21538; ABI21527;ABI20856; ABG47837; ABF47668 1999 ACR15312; ACF41886; ABK40014; ABJ166174 2.1 0.3 low p < .01, prev p < .20 2000 Q82571; AAF99677; AAF99676;AAX56539; ABV45857; ABU80317; 82 2.0 0.1 low p < .001, ABU80306;ABS49995; ABS49984; ABR28809; ABR28787 prev p < .40 ABR28776; ABR15926;ABR15915; ABR15904; ABR15893; ABP49390; ABP49313; ABP49225; ABO44054;ABM22034; ABL67217; ABL67195; ABK79978; ABK40058; ABK40047; ABK40036;ABJ53523; ABJ53457; ABJ16738; ABJ16727; ABJ16650; ABJ09335; ABI95302;ABI95225; ABG88561; ABG88550; ABG80191; ABG80180; ABG67488; ABG48057;ABG37370; ABF47899; ABF47888; ABF47877; ABG47826; ABG47815; ABE11676;ABE11665; ABD95039; ABD95028; ABD95017; ABD95006; ABD94995 ABD94984;ABD94973; ABD94764; ABD78046; ABD78035; ABD78024; ABD78013; ABD78002;ABD77991; ABD77980; ABD77969; ABD77958; ABD77947; ABD77936; ABD77925;ABD77738; ABD77727; ABD77716; ABD63071; ABD61548; ABD61526; ABD60908;ABD60897; ABD60886; ABD60875; ABD60864; ABA08505; ABA08494; 2001ABR28853; ABR28842; ABO38337; ABO38326; ABO38051; ABO38040; 116 2.0 0.1low p < .001, ABO38029; ABO32967; ABO32956; ABN51151; ABN51085; prev p >.50 ABM66872; ABJ09159; ABG67499; ABG37403; ABG37392; ABG26953;ABF82948; ABF82937; ABF82926; ABF82915; ABF82904; ABF82893; ABF82882;ABF82871; ABF82860; ABF82849; ABF82838; ABF82827; ABF47679; ABF47580;ABF47569; ABG37128; ABF82692; ABF82681; ABF82670; ABF47591; ABE12292;ABE11864; ABE11853; ABE11842; ABE11831; ABE11820; ABE11742; ABE11731;ABE11720; ABE11709; ABE11698; ABE11687; ABD95336; ABD95325; ABD95314;ABD95303; ABD95292; ABD95281; ABD95270; ABD95259; ABD95248; ABD95237;ABD95226; ABD95215; ABD95204; ABD95193; ABD95182; ABD95171; ABD95160;ABD95149; ABD95138; ABD95127; ABD95116; ABD95105; ABD95094; ABD95083;ABD95072; ABD95061; ABD95050; ABD94819; ABD94808; ABD94797; ABD94786;ABD78101; ABD78090; ABD78079; ABD78068; ABD60919; ABC86245; ABC40541;ABB02822; ABA87239; ABA87099; ABC02285; ABB82202; ABB80053; ABB79998;ABB79987; ABB53715; ABB02944; ABB02932; ABB02921; ABB02833; ABA87053;ABA43197; ABA42583; ABA42332; ABA42266; ABA42244; ABA18045; ABA12726;ABA08527; ABA08472; AAZ85134; AAZ83307; AAZ79612; AAZ38635; AAK18014;AAK18013 2002 ACR15334; ACR15323; ACR15224; ABA87088; ABB82224; AAZ832616 2.0 0.0 low p < .001, prev p < .02 2003 AAO88267; ABN51096; ABM67059;ABD60787; ABD15523; ABC41722; 23 2.0 0.1 low p < .001, ABB03131;ABA87065; AAZ83985; ABB82213; ABB80111; prev p > .50 ABB53748; ABB03153;ABB02811; ABB02800; ABA42255; ABA18153; ABA12737; ABA12716; ABA12704;ABA08483; ABK40003; CAD58687 2004 ABC42758 1 2.0 0.0 prev p > .50 2005ABR28908; ABP49401; ABO32978; ABO32686; ABK40697; ABJ16694; 20 2.4 3.0low p < .10, ABJ16683; ABJ16672; ABJ16661; ABJ09192; ABI92387; prev p <.40 ABI30573; ABI22156; ABI21241; ABI21230; ABI21219; ABI21208;ABI21197; ACG50704; P0C0U1 2006 ABD59820; ABD59818; ABD59816; ABB86941;ABB86958; ABB86955; 27 2.1 0.7 low p < .001, ABB86954; ABB86950;ABB86931; ABB86901; ABB86881; prev p > .50 ABB86871; ACO94812; ACI26458;ABX58687; ABX58247; ABW71302; ABV29565; ABV29554; ABV29543; ABK79967;ACN72626; ABB86921; ABB86911; ABB86891 ABG88887 2007 Q1WP01; Q3HM40;ABS00317; ACR15202; ACN43000; ACN42989; 320 2.1 1.1 low p < .001,ACN33109; ACN33098; ACN32845; ACN32834; ACN32823; prev p > .50 ACN32812;ACN32801; ACL12071; ACF41688; ACD56288; ACD56132; ACD56121; ACC61994;ACC61983; ACC61972; ACA96527; ACA24532; ACA24521; ABY81423; ABY81412;ABY81401; ABY81390; ABY81368; ABY51267; ABY51256; ABY51245; ABY51201;ABY51190; ABY51179; ABY51168; ABY51157; ABY51146; ABY51124; ABY51113;ABY51102; ABY51091; ABY51080; ABY51069; ABY51058; ABY51047; ABY51025;ABX58720; ABX58709; ABX58698; ABX58643; ABX58632; ABX58621; ABX58610;ABX58599; ABX58588; ABX58577; ABX58566; ABX58555; ABX58544; ABX58533;ABX58522; ABX58511; ABX58500; ABX58489; ABX58478; ABX58467; ABX58456;ABX58445; ABX58434; ABX58423; ABX58412; ABX58401; ABX58390; ABX58379;ABX58368; ABX58357; ABX58346; ABX58335; ABX58324; ABX58313; ABX58302;ABX58291; ABX58280; ABX58269; ABX58258; ABW91644; ABW91633; ABW91622;ABW91611; ABW91600; ABW91589; ABW91578; ABW91567; ABW91545; ABW91534;ABW91523; ABW91512; ABW91501; ABW91490; ABW91479; ABW91468; ABW91457;ABW91435; ABW91424; ABW91413; ABW91391; ABW91380; ABW91369; ABW91358;ABW91347; ABW91336; ABW91325; ABW91314; ABW91303; ABW91292; ABW91281;ABW91226; ABW86614; ABW86560; ABW86549; ABW86538; ABW86527; ABW86516;ABW86505; ABW86494; ABW86483; ABW86472; ABW86461; ABW86450; ABW86439;ABW86428; ABW86417; ABW86406; ABW86395; ABW86384; ABW86373; ABW86362;ABW86351; ABW86340; ABW86329; ABW71478; ABW71467; ABW71456; ABW71445;ABW71434; ABW71423; ABW71412; ABW71401; ABW71390; ABW71379; ABW71368;ABW71346; ABW71335; ABW71324; ABW71313; ABW40683; ABW40672; ABW40650;ABW40628; ABW40617; ABW40606; ABW40584; ABW40573; ABW40562; ABW40551;ABW40540; ABW40529; ABW40518; ABW40507; ABW40496; ABW40485; ABW40474;ABW40463; ABW40452; ABW40441; ABW40430; ABW40419; ABW40408; ABW40397;ABW40375; ABW40364; ABW40353; ABW40342; ABW40320; ABW40309; ABW40298;ABW40287; ABW40265; ABW40243; ABW40232; ABW40221; ABW40210; ABW40188;ABW40166; ABW40155; ABW40133; ABW40122; ABW40111; ABW40100; ABW40078;ABW40067; ABW40056; ABW40045; ABW40023; ABW40012; ABW40001; ABW39990;ABW39979; ABW39968; ABW39957; ABW39935; ABW39924; ABW39913; ABW39902;ABW39891; ABW39869; ABW39858; ABW39847; ABW39836; ABW39825; ABW39814;ABW39785; ABW36308; ABW36297; ABW36286; ABW36275; ABW36264; ABW36253;ABW36242; ABW36231; ABW36220; ABW36209; ABW36198; ABW36187; ABV82559;ABV45967; ABV45956; ABV45945; ABV45934; ABV45923; ABV45901; ABV45890;ABV45879; ABV30632; ABV30621; ABV30610; ABV30599; ABV30588; ABV30577;ABV30566; ABV30555; ABV30544; ABV30533; ABV30511; ABV30500; ABV30467;ABV30379; ABV30368; ABV30357; ABV30346; ABV30335; ABV30324; ABV30313;ABV30302; ABV30291; ABV30203; ABV30192; ABV30181; ABV30170; ABV30159;ABV30148; ABV30137; ABV30115; ABV30104; ABV30093; ABV30060; ABV30049;ABV30038; ABV30027; ABV30016; ABV30005; ABV29994; ABV29983; ABV29972;ABV29961; ABV29950; ABV29928; ABV29895; ABV29884; ABV29873; ABV29862;ABV29851; ABV29840; ABV29807; ABV29796; ABV29785; ABV29774; ABV29763;ABV29752; ABV29741; ABV29708; ABV29697; ABV29686; ABV29675; ABV29664;ABV29653; ABV29642; ABV29620; ABV29609; ABV29587; ABV29576; ACN72614;ACR61674; ACR61663; P0C574; Q20MH0; Q1WP00; ABS00328; Q8JSD9 2008ACP20229; ACP20218; ABV01075; ACR15521; ACR15510; ACR15499; 49 2.0 0.2low p < .001, ACR15488; ACR15477; ACR15466; ACR15455; ACR15444; prev p <.04 ACR15433; ACR15378; ACR15367; ACR15345; ACR15279; ACR15268;ACR15246; ACQ65766; ACP44233; ACP44222; ACP44211; ACP44200; ACO95423;ACO95412; ACO95401; ACO95390; ACO95379; ACO94878; ACO94867; ACO94713;ACO36405; ACN33153; ACN33131; ACN32520; ACN32509; ACL12159; ACI26447;ACF54595; ABV01079; ABV01076; ABV01074; ABV01073; ABV01070; ABV01069;ABV01068; ACR58560; ACR58549 2009 A4GCI3; A4GCK5; A4GBY5; B3EUR4;Q0HD52; ACP41103; ACR47013; 177 3.2 3.7 low p < .001, ACR08608;ACR55002; ACR08503; ACR09394; ACR09393; prev p < .001 ACR09392;ACR09391; ACR08590; ACR08588; ACR08586; ACR08585; ACQ99679; ACQ99678;ACQ99677; ACQ99676; ACQ99675; ACQ83306; ACQ76409; ACQ76378; ACQ76372;ACQ76357; ACQ76349; ACQ76320; ACQ76306; ACQ76296; ACQ76289; ACQ63280;ACQ63255; ACQ63247; ACQ63231; ACQ55362; ACP44176; ACP44169; ACP44165;ACP41958; ACP41941; ACP41933; ACR46669; ACR46660; ACR20067; ACR78576;ACR67252; ACR67242; ACR54992; ACR54982; ACR54972; ACR54962; ACR52496;ACR52486; ACR52476; ACR52466; ACR52456; ACR52446; ACR52436; ACR52426;ACR52416; ACR39501; ACR39461; ACR52396; ACR52386; ACR52376; ACR51073;ACR51063; ACR51053; ACR51043; ACR51033; ACR51023; ACR51013; ACR51003;ACR40306; ACR40396; ACR40386; ACR40376; ACR40366; ACR40356; ACR40346;ACR40336; ACR40326; ACR40316; ACR40296; ACR39491; ACR39481; ACR39471;ACR39451; ACR39441; ACR39431; ACR39421; ACR39411; ACR39401; ACR39362;ACR38881; ACR18922; ACR15356; ACR10219; ACR10218; ACR10194; ACR08589;ACR08587; ACR08584; ACQ84475; ACQ84465; ACR08467; ACR08457; ACR08447;ACR08437; ACR08427; ACQ73409; ACQ73411; ACQ73410; ACQ89953; ACQ89952;ACQ89951; ACQ89950; ACQ89949; ACQ89948; ACO94845; ACO94834; ACR54044;ACR49313; ACR49312; ACR49311; ACR49310; ACR49309; ACR49308; ACR49307;ACR49306; ACR49305; ACR15750; ACR15613; ACR77506; ACR77496; ACR77486;ACR77476; ACR77466; ACR77456; ACR77446; ACR67121; ACR67120; ACR67119;ACR67118; ACR56458; ACR56448; ACR56438; ACR56428; ACR56418; ACR56408;ACR56398; ACR56388; ACR38797; ACR38796; ACR38795; ACR78469; ACR54049;A4GCI4; A4GCK6; A4GBY6; B3EUR5; Q0HD51; A3DRP9; A8C8X2; A4K152; A4U7B5;A4U6W1; A4GCL7; A4GCJ5; A4GCM8; A8C8K3; B4URE5; Q289L8; Q07FH6

Example 5 Double Differentiation of Infectivity and Lethality in 2009Global Outbreak of H1N1 Influenza Virus Through Sep. 23, 2009

The infectivity and lethality of the H1N1 influenza virus causing the2009 global outbreak of H1N1 influenza virus was differentiated byanalyzing the Replikin Counts of sequences of the hemagglutinin proteinarea and pB1 gene area of isolates of H1N1 publicly available atwww.pubmed.com from 2001 through Sep. 23, 2009.

The Replikin Count (number of Replikin sequences per 100 amino acidresidues of a sequence) of the publicly available hemagglutininsequences and the publicly available pB1 gene area sequences wereanalyzed using FLUFORECAST® software (Replikins, Ltd., Boston, Mass.).The results of the analysis are provided below in Table 7.

TABLE 7 H1N1 Influenza Virus Infectivity and Lethality in Humans 1 Jan.2001 through 23 Sep. 2009 pB1 Gene Area Mean Hemagglutinin Annual MeanAnnual Hemagglutinin Replikin pB1 Gene Area Replikin Count StandardSequences Count Standard Sequences Date (Infectivity) Deviation Analyzed(Lethality) Deviation Analyzed 2001 4.3 2 144 2 0.1 122 2002 3.5 1.9 622 0.1 4 2003 4.8 1.3 88 2 0 25 2004 5 3.1 15 2 0 6 2005 5.2 2.7 68 1.80.4 19 2006 5 2.3 102 2.2 0.7 27 2007 6 1.6 537 2.1 1.1 318 30 JUN 086.7 1.2 320 2 0.2 41 31 DEC 08 7 1.3 491 2 0.5 118 30 APR 09 10 2.4 293.7 4.5 155 6 JUN 09 9.7 2.4 357 3.3 4 13 JUN 09 10 1.9 3 3.6 15 JUN 099.7 2.4 3 3.5 16 JUN 09 9.7 1.8 3 3.5 203 20 JUN 09 9.8 1.8 415 3 3.5203 21 JUN 09 9.8 1.8 425 3 3.5 254 23 JUN 09 9.8 1.8 425 2.8 3.2 209 26JUN 09 9.9 1.8 532 3 3.5 226 29 JUN 09 9.9 1.6 553 2.9 3.3 226 30 JUN 099.9 1.6 553 2.9 3.3 230 2 JUL 09 9.9 1.6 559 2.6 2.8 231 4 JUL 09 10 1.6563 2.9 3.3 223 6 JUL 09 10 1.6 519 2.9 3.3 222 8 JUL 09 10 1.6 585 2.93.4 254 10 JUL 09 9.6 1.3 627 2.7 3 254 12 JUL 09 9.6 2.2 627 2.8 3.2265 14 JUL 09 9.6 2.1 652 2.8 3.2 261 18 JUL 09 9.6 2.1 652 2.8 3.1 27522 JUL 09 9.6 2.1 654 2.7 3 295 24 JUL 09 9.7 2.1 654 2.7 3.1 295 25 JUL09 9.7 2.1 683 2.7 3 326 3 AUG 09 9.6 2.1 747 2.6 2.8 319 6 AUG 09 9 2.8820 2.6 2.8 345 8 AUG 09 9 2.8 820 2.6 2.7 340 10 AUG 09 9.6 2.1 771 2.62.7 345 12 AUG 09 9.6 2.1 777 2.6 2.7 373 14 AUG 09 9.6 2.1 804 2.5 2.6373 15 AUG 09 9.6 2.1 812 2.5 2.6 373 16 AUG 09 9.5 2.1 803 2.5 2.6 37317 AUG 09 9.5 2.1 812 2.5 2.6 373 18 AUG 09 9.6 2.1 812 2.5 2.6 371 20AUG 09 9.6 2.1 810 2.1 2.7 376 22 AUG 09 9.6 2.1 817 2.5 2.6 378 27 AUG09 9.6 2.1 820 2.5 2.6 476 28 AUG 09 9.6 2.1 855 2.6 2.8 408 30 AUG 099.6 2.1 855 2.5 2.5 408 1 SEP 09 9.6 2 834 2.5 2.5 395 3 SEP 09 9.6 2.1841 2.5 2.6 422 4 SEP 09 9.6 2.1 852 2.3 2.5 422 7 SEP 09 9.6 2.1 8692.5 2.5 422 10 SEP 09 9.6 2.1 870 2.5 2.5 457 12 SEP 09 9.6 2 932 2.42.4 532 13 SEP 09 9.6 2 932 2.4 2.2 532 14 SEP 09 9.7 1.9 992 2.4 2.2532 15 SEP 09 9.6 1.9 1,006 2.4 2.2 532 16 SEP 09 9.6 1.9 1,013 2.4 2.2531 17 SEP 09 9.6 1.9 1,005 2.4 2.2 516 18 SEP 09 9.6 1.9 992 2.4 2.3527 19 SEP 09 9.7 1.9 1,023 2.4 2.2 504 20 SEP 09 9.7 1.8 1,003 2.4 2.3504 21 SEP 09 9.7 1.8 1,003 2.4 2.3 521 22 SEP 09 9.7 1.9 1,022 2.4 2.3506 23 SEP 09 9.6 1.9 994 2.3 1.9 118

As may be seen from the data in Table 7 (and the illustration of thedata in FIG. 5), analysis of the Replikin Counts of hemagglutininprotein area sequences and the pB1 gene area sequences for isolates ofH1N1 from 2001 through Sep. 23, 2009 reveal that Replikin Counts in thehemagglutinin protein area and Replikin Counts in the pB1 gene area areindependent from one another in isolates from a given year, given month,or given day and Replikin Counts in these areas of the genome trend indirections that are independent from one another. As may further be seenin Table 7 and FIG. 5, mean annual Replikin Counts in the hemagglutininprotein area trended upward from 2001 through 2009 while mean annualReplikin Counts in the pB1 gene area stayed about the same with a smallspike in mean annual Replikin Count in 2005 that was not statisticallysignificant (p<0.40) and a notable spike in mean annual Replikin Countin 2009 that is statistically significant (p<0.001) followed by a slowdecreasing trend in Replikin Count in the pB1 gene area from Apr. 30,2009 through Sep. 23, 2009.

By determining mean Replikin Counts among isolates of H1N1 havinghemagglutinin and pB1 gene area sequences available at www.pubmed.com,the applicants published a warning on Apr. 7, 2008 that the H1N1 virushad arisen as the most likely candidate for the next pandemic. Seehttp://www.replikins.com/release.html#article18. This warning waspublished one year before the current pandemic outbreak of H1N1. Theanalysis that led to the warning was undertaken using FluForecast®software to analyze publicly available sequences from H1N1 influenzavirus in humans. In following changes in Replikin Count in thehemagglutinin protein area, the applicants discovered that the ReplikinCount, which had been increasing since 2001, had reached a mean level ofseven Replikin sequences per one hundred amino acids, a concentrationthat had been observed previously only in isolates from the H1N1pandemic of 1918. Following this warning, H1N1 outbreaks were reportedin Mexico and California in the first three months of 2009. Theoutbreaks then expanded into the present 2009 H1N1 pandemic. Since April2009, the applicants have provided advance information on the changingvirus structure using their FluForecast® software methods. See FIG. 5.The advance information has predicted the clinical course of the H1N1pandemic.

FIG. 5 illustrates analysis of all data published on PubMed forconcentrations of Replikin sequences in the virus genomes. The dates inFIG. 5 represent the publication dates of each specimen sequence atwww.pubmed.com. Publication dates generally reflect one to four monthsdelay from the date a specimen is actually collected. This timedifference represents time taken for sequence analysis, review, andpublication. As one of ordinary skill in the art would understand, morereal-time analysis of concentrations of Replikin sequences would beexpected to improve the predictive and analytical capacity of knownReplikin concentrations. Closer-in-time analysis could be realized ifthe time for sequence analysis and publication were shortened.

Additionally, greater numbers of publicly reported isolates would beexpected to provide improved analysis of mean Replikin Counts. Thenumber of specimens publicly available for analysis from 2001 through2008 was a total of 855 specimens. Through Sep. 23, 2009, the number ofpublicly available specimens in 2009 alone has been 1,555. As thesenumbers increase, Replikin Count analysis would be expected to improvein accuracy within time periods and within specific regions.

As may be seen from FIG. 5, elevated Replikin Counts in thehemagglutinin protein area (or Infectivity Gene) of 2009 isolates hascontinued to remain high, with Replikin Counts consistently above nineReplikin sequences per one hundred amino acid residues in thehemagglutinin protein area. The data in FIG. 5, therefore, predictedthat H1N1 infection would continue above seasonal norms in the summer of2009 in the Northern Hemisphere and in the winter of 2009 in theSouthern Hemisphere. Despite this prediction, many public healthofficials continued to expect that the 2009 H1N1 pandemic would beinterrupted in the northern summer. This expectation of public healthofficials was nevertheless contradicted by a high level of infectionsthroughout the 2009 summer in the U.S., U.K., China and many otherNorthern Hemisphere countries. The pandemic also continued unabated inthe Southern Hemisphere in its 2009 winter season.

As may be seen from FIG. 5, a peak in Replikin Count in both thehemagglutinin protein area and in the pB1 gene area is observed betweenDecember 2008 and April 2009. As would be expected with peaks inReplikin Count, the December 2008 to April 2009 peak was followed (twoto six months later) in the U.S. by a peak in pediatric deaths in Juneof 2009. See CDC FluView, Week 36 ending Sep. 12, 2009 available athttp://www.cdc.gov/flu/weekly/. To our knowledge, prior to the discoveryof Replikin sequences, no virus structure had been reported thatquantitatively correlated with or predicted virus outbreaks or theclinical course of virus outbreaks. FIG. 5 demonstrates a quantitativecorrelation with and prediction of both outbreaks and their clinicalcourse.

FIG. 5 shows that for the H1N1 Infectivity Gene (hemagglutinin proteinarea in white), the Mean Replikin Count increased from 4.3 (+/−2) in2001 to 6.7 (+/−1.2) in 2008 (p<0.001). At that time, the applicantspublished their warning that H1N1 was the leading candidate for apandemic. The Mean Replikin Count then continued to increase 43% to amean count of 10 by April 2009. At that point, the clinical H1N1outbreak in Mexico and California was reported. By June 2009, the WorldHealth Organization stated that the outbreak had sufficiently spreadglobally to be declared a pandemic.

As of September 2009, the Infectivity Gene Count (hemagglutinin inwhite) remains elevated, decreasing only 3% in its mean since the highin April 2009, thus giving no significant sign of abatement (as yet) inthe current pandemic. If the Replikin Count were to decreasesignificantly, an abatement such as that which occurred in SARS would beexpected. In the SARS outbreak of 2003, a sharp drop in the ReplikinCount of the spike protein in 2003 signaled the abrupt end of theclinical outbreak. See U.S. application Ser. No. 12/010,027, filed Jan.18, 2008, FIG. 9.

FIG. 5 also shows that for the H1N1 Lethality Gene (pB1 gene area, inblack), the Mean Replikin Count between 2001 to 2008, despite someactivity, did not increase significantly (in contrast to the InfectivityGene—hemagglutinin protein area). However, the Standard Deviation of theMean (SD) in the pB1 gene area (represented by capped lines) increasedfive-fold between 2001 and December 2008 and forty-five fold between2001 and April 2009. An increase in standard deviation of mean ReplikinCount indicates that some viruses in a virus population have highReplikin Counts and are engaging in high replication rates.

As may be seen in FIG. 5, mean Replikin Count in the pB1 gene area ofH1N1 isolates has gradually decreased by 38% from its high in April 2009through to Sep. 23, 2009 (p<0.001). However, the mean Replikin Count isstill 15% higher, and the standard deviation of the mean is still 19times greater, than the level of Replikin Count seen in 2001, which maybe considered a “resting rate” for purposes of FIG. 5. These higherReplikin Counts indicate that there are still active individual viruseswithin the currently circulating H1N1 virus population that containincreased Replikin Counts in their Lethality Genes. The overall trendseen in FIG. 5 since April 2009, however, is clearly towards a return tothe lower “resting” Replikin Count of about two, which predominated from1980 to 2008 (or less than two, which predominated from 1934 to 1979).These low Replikin Counts from 1934 to 2008 were accompanied by lowclinical H1N1 lethality.

The recent increase in the Replikin Count of the Replikin InfectivityGene of H1N1 (which gave warning of the H1N1 pandemic of 2009) togetherwith the current statistically significant decline in the Replikin Countof the Lethality Gene (which was followed by a sharp drop in H1N1pediatric mortality since June 2009) raise the possibility that,although high infectivity will persist, there is no indication atpresent that a high mortality rate is to be expected. Nevertheless, asthe Replikin Count is further monitored, the status of the infectivityand lethality of the current H1N1 pandemic (as determined by ReplikinCount) may change at any time, as the lethality gene Replikin Count didat the beginning of 2009.

Example 6 Replikin Count by Year for H1N1 from 1933 Through 2008

The applicants reviewed publicly available pB1 gene area sequences fromisolates of H1N1 influenza virus isolated between 1933 and 2000 atwww.pubmed.com. The data is provided in Table 8 below. After a highReplikin Count in the pB1 gene area of influenza isolates in 1933(associated with the H1N1 outbreak of that year), the data demonstrate aremarkable consistency from 1934 through 1980 (Replikin Counts generallybelow two) and a remarkable consistency from 1981 through 2000 (ReplikinCounts generally around two). (1933 was the last significant outbreak ofH1N1 prior to the present pandemic. The small and limited outbreak of1976, was marked by a Replikin Count of 1.9+/−0.2, and never developedfurther, as would be expected from the low Replikin Count, either in itsCount or clinically. Had the Replikins been known at that time, thehurried vaccination of millions of people because of the fear of anotherH1N1 pandemic might have been avoided.) This consistency in ReplikinCount in the pB1 gene area continued through 2008. See FIG. 5. It wasbroken, however, beginning in December of 2008 when it rose from 2 to3.7 between December 2008 and April 2009. See Table 7 above and FIG. 5.This significant rise in the Replikin Count of the pB1 gene area(Lethality Gene) corresponds to the outbreak of the present pandemic.

TABLE 8 H1N1 Annual Mean Replikin Count in pB1 Gene Area Mean No. ofReplikin Isolates Count Year PubMed Accession Number-Replikin Count peryear per year S.D. Significance 1933 ABD77804 18 ACV49542 18 ACF54606 18ABF47963 18 4 2.4 0.0 low p < 0.001 1934 ACV49553 12 ACF41842 12ABD77683 12 3 1.6 0.0 low p < 0.001, prev p < 0.001 1935 ABD62789 14ABO38392 11 ABN59420 12 3 1.6 0.2 low p < 0.20, prev p > 0.50 1940ABI20834 11 1 1.5 0.0 1940 ABI20834 1 1.5 0.0 1942 ABD62850 9 1 1.2 0.01943 ABO38381 11 ABO38062 11 2 1.5 0.0 prev p < 0.001 1945 ABP49335 11 11.5 0.0 1946 ABD79120 11 ACV49564 14 2 1.7 0.3 low p > 0.50, prev p >0.50 1947 ABD77815 11 ACV49575 11 2 1.5 0.0 prev p < 0.40 1948 ABN5940912 1 1.6 0.0 prev p < 0.001 1949 ABN59442 12 1 1.6 0.0 1950 ABD61743 12ABP49324 12 2 1.6 0.0 low p < 0.001 1951 ABR15816 12 ABQ44479 12ABQ01319 12 ABP49489 12 4 1.6 0.0 low p < 0.001 1954 ABD60974 11ABO52288 11 2 1.5 0.0 prev p < 0.001 1957 ABD15267 12 1 1.6 0.0 prev p <0.001 1976 ACU80022 14 ACQ99829 14 ABV45846 16 ABQ44402 14 3 1.9 0.2 lowp < 0.02, prev p < 0.05 1977 ABD95358 11 ABD60941 11 ABO44142 12 4 1.50.1 low p < 0.30, prev p < 0.002 1978 ABY81357 14 ABP49456 8 ABP49346 14ABO38073 8 14 1.4 0.3 low p < 0.40, ABO33000 8 ABO32989 11 ABN59431 8ABK79956 11 prev p < 0.20 ABG26821 11 ABF47745 11 ABF47734 11 ABF4772311 ABF47712 11 ABF47701 11 1979 ABW36319 14 ABQ01330 14 ABN50764 14 31.8 0.0 low p < 0.001, prev p < 0.001 1980 ABO38370 15 ABO33017 15ABF47756 15 3 2.0 0.0 low p < 0.001, prev p < 0.001 1981 ABO52266 15 12.0 0.0 prev p > 0.50 1982 ABD95347 18 ABO52805 15 2 2.2 0.3 low p <0.10, prev p < 0.50 1983 ABW91193 15 ABO38348 15 ABO37996 15 ABO33033 1547 2.0 0.1 low p < 0.001, ABN50925 15 ABN50908 18 ABM66894 15 ABM6691615 prev p < 0.40 ABM66905 15 ABM22243 15 ABM22232 15 ABM22221 15ABM22210 15 ABM22199 15 ABM22188 15 ABM22177 15 ABM22166 15 ABL67272 15ABL67261 15 ABK80055 15 ABK80044 15 ABK80033 15 ABK40609 15 ABK40598 15ABK40587 15 ABK40576 15 ABK40565 15 ABK40554 15 ABK40543 15 ABK40518 15ABI92310 18 ABI30386 15 ABI20867 15 ABG88352 18 ABG88341 18 ABF47833 15ABF47778 15 ABG79960 15 ABF47855 15 ABF47844 15 ABF47767 15 ABF47800 15ABG26843 15 ABG26832 15 ABF47822 15 ABF47811 15 ABF47789 15 1984ABP49357 15 ABO38414 15 2 2.0 0.0 low p < 0.001, prev p < 0.04 1986P03430 18 P03431 12 ABP49368 15 ABO44131 15 ABO38403 7 2.0 0.3 low p <0.001, 15 ABM22254 15 P03427 18 prev p > 0.50 1987 ACV49674 15 ABQ4442415 ABN50948 15 ABN50936 15 4 2.0 0.0 low p < 0.001, prev p > 0.50 1988ABU80408 16 1 2.1 0.0 prev p < 0.001 1989 ACL12269 15 ACK99451 15 2 2.00.0 low p < 0.001, prev p < 0.001 1990 P16512 20 P16510 17 P16514 16P18882 14 P16502 12 5 2.1 0.4 low p < 0.02, prev p > 0.50 1991 ABD6096315 ACQ84485 15 ACF41941 15 ACF41930 12 4 1.9 0.2 low p < 0.02, prev p <0.30 1993 AAA43643 16 AAA43641 12 AAA43640 20 AAA43639 17 6 2.1 0.4 lowp < 0.005, AAA43582 18 AAA43581 14 prev p < 0.20 1995 ACK99473 15ACF41875 15 ABG88330 15 ABG26799 15 24 2.0 0.1 low p < 0.001, ABF4764615 ABJ53446 15 ABI92321 15 ABI30375 15 prev p < 0.20 ABI20878 15ABI20845 15 ABG88319 15 ABG88308 15 ABF47635 15 ABG47848 15 ABG26788 15ABF47613 15 ABE26999 15 ABE12040 15 ABE11968 13 ABE11930 15 ABE11908 15ABE11897 15 ABE11886 15 ABE11875 15 1996 ABO52233 15 ABO38018 15ABN51074 15 ABN50981 15 27 2.0 0.1 low p < 0.001, ABN50970 15 ABN5095915 ABF47657 15 ABM22298 15 prev p > 0.50 ABM22287 15 ABM22276 15ABM22265 15 ABJ53512 15 ABJ53501 15 ABI95291 15 ABI95280 15 ABI95269 15ABI95258 15 ABI93036 15 ABI21582 15 ABI21571 15 ABI21560 15 ABI21549 15ABI21538 15 ABI21527 15 ABI20856 11 ABG47837 15 ABF47668 15 1999ACR15312 19 ACF41886 15 ABK40014 15 ABJ16617 15 4 2.1 0.3 low p < 0.01,prev p < .20 2000 Q82571 11 AAF99677 17 AAF99676 17 ABV45857 15 81 2.00.1 low p < 0.001, ABU80317 15 ABU80306 15 ABS49995 15 ABS49984 15 prevp < 0.40 ABR28809 15 ABR28787 15 ABR28776 15 ABR15926 15 ABR15915 15ABR15904 15 ABR15893 15 ABP49390 15 ABP49313 18 ABP49225 15 ABO44054 15ABM22034 15 ABL67217 15 ABL67195 15 ABK79978 15 ABK40058 15 ABK40047 15ABK40036 15 ABJ53523 15 ABJ53457 15 ABJ16738 15 ABJ16727 15 ABJ16650 15ABJ09335 17 ABI95302 15 ABI95225 15 ABG88561 15 ABG88550 15 ABG80191 15ABG80180 15 ABG67488 15 ABG48057 15 ABG37370 19 ABF47899 15 ABF47888 15ABF47877 15 ABG47826 15 ABG47815 15 ABE11676 17 ABE11665 15 ABD95039 15ABD95028 15 ABD95017 15 ABD95006 15 ABD94995 15 ABD94984 17 ABD94973 15ABD94764 15 ABD78046 12 ABD78035 15 ABD78024 15 ABD78013 15 ABD78002 15ABD77991 15 ABD77980 15 ABD77969 15 ABD77958 15 ABD77947 15 ABD77936 15ABD77925 15 ABD77738 15 ABD77727 15 ABD77716 18 ABD63071 15 ABD61548 15ABD61526 15 ABD60908 15 ABD60897 15 ABD60886 15 ABD60875 15 ABD60864 15ABA08505 15 ABA08494 15 2001 ABR28853 15 ABR28842 15 ABO38337 15ABO38326 15 116 2.0 0.1 low p < 0.001, ABO38051 15 ABO38040 17 ABO3802915 ABO32967 15 prev p > 0.50 ABO32956 15 ABN51151 15 ABN51085 15ABM66872 15 ABJ09159 15 ABG67499 15 ABG37403 15 ABG37392 15 ABG26953 15ABF82948 15 ABF82937 15 ABF82926 15 ABF82915 18 ABF82904 15 ABF82893 15ABF82882 15 ABF82871 15 ABF82860 15 ABF82849 15 ABF82838 15 ABF82827 15ABF47679 15 ABF47580 15 ABF47569 15 ABG37128 15 ABF82692 15 ABF82681 15ABF82670 15 ABF47591 18 ABE12292 15 ABE11864 15 ABE11853 15 ABE11842 15ABE11831 15 ABE11820 15 ABE11742 15 ABE11731 15 ABE11720 15 ABE11709 15ABE11698 15 ABE11687 15 ABD95336 15 ABD95325 15 ABD95314 15 ABD95303 15ABD95292 15 ABD95281 15 ABD95270 15 ABD95259 15 ABD95248 15 ABD95237 15ABD95226 15 ABD95215 15 ABD95204 15 ABD95193 15 ABD95182 15 ABD95171 15ABD95160 15 ABD95149 15 ABD95138 15 ABD95127 15 ABD95116 15 ABD95105 15ABD95094 15 ABD95083 15 ABD95072 15 ABD95061 15 ABD95050 15 ABD94819 15ABD94808 15 ABD94797 15 ABD94786 15 ABD78101 15 ABD78090 15 ABD78079 15ABD78068 15 ABD60919 18 ABC86245 15 ABC40541 15 ABB02822 15 ABA87239 15ABA87099 15 ABC02285 15 ABB82202 15 ABB80053 15 ABB79998 15 ABB79987 15ABB53715 15 ABB02944 15 ABB02932 15 ABB02921 15 ABB02833 15 ABA87053 15ABA43197 15 ABA42583 15 ABA42332 18 ABA42266 15 ABA42244 15 ABA18045 15ABA12726 15 ABA08527 18 ABA08472 15 AAZ85134 15 AAZ83307 15 AAZ79612 15AAZ38635 15 AAK18014 14 AAK18013 15 2002 ACR15334 15 ACR15323 15ACR15224 15 ABA87088 15 6 2.0 0.0 low p < 0.001, ABB82224 15 AAZ83261 15prev p < 0.02 2003 AAO88267 15 ABN51096 15 ABM67059 15 ABD60787 15 232.0 0.1 low p < 0.001, ABD15523 15 ABC41722 19 ABB03131 15 ABA87065 15prev p > 0.50 AAZ83985 15 ABB82213 15 ABB80111 15 ABB53748 15 ABB0315314 ABB02811 15 ABB02800 15 ABA42255 15 ABA18153 15 ABA12737 15 ABA1271615 ABA12704 15 ABA08483 15 ABK40003 12 CAD58687 6 2004 ABC42758 15 1 2.00.0 prev p > 0.50 2005 ABR28908 15 ABP49401 15 ABO32978 15 ABO32686 8 202.4 3.0 low p < 0.10, ABK40697 8 ABJ16694 15 ABJ16683 15 ABJ16672 15prev p < 0.40 ABJ16661 15 ABJ09192 8 ABI92387 15 ABI30573 15 ABI22156 15ABI21241 15 ABI21230 8 ABI21219 15 ABI21208 15 ABI21197 15 ACG50704 15P0C0U1 13 2006 ABD59820 19 ABD59818 15 ABD59816 15 ABB86941 15 25 2.10.7 low p < 0.001, ABB86958 16 ABB86955 13 ABB86954 17 ABB86950 14 prevp > 0.50 ABB86901 15 ABB86871 14 ACO94812 15 ACI26458 15 ABX58687 15ABX58247 15 ABW71302 15 ABV29565 15 ABV29554 15 ABV29543 17 ABK79967 15ACN72626 20 ABB86921 14 ABB86911 12 ABB86891 16 ABG88887 14 2HN8_A 22007 Q3HM40 14 Q1WP01 11 ABS00317 15 ACU80176 15 318 2.1 1.1 low p <0.001, ACU80000 15 ACR61674 15 ACR61663 15 ACR15202 15 prev p > 0.50ACN43000 17 ACN42989 17 ACN33109 15 ACN33098 15 ACN32845 15 ACN32834 15ACN32823 15 ACN32812 15 ACN32801 15 ACL12071 15 ACF41688 15 ACD56288 15ACD56132 19 ACD56121 15 ACC61994 15 ACC61983 15 ACC61972 15 ACA96527 15ACA24532 15 ACA24521 15 ABY81423 15 ABY81412 15 ABY81401 17 ABY81390 15ABY81368 15 ABY51267 15 ABY51256 15 ABY51245 15 ABY51201 15 ABY51190 15ABY51179 15 ABY51168 15 ABY51157 15 ABY51146 17 ABY51124 15 ABY51113 15ABY51102 15 ABY51091 17 ABY51080 15 ABY51069 15 ABY51058 15 ABY51047 15ABY51025 15 ABX58720 15 ABX58709 17 ABX58698 15 ABX58643 15 ABX58632 15ABX58621 15 ABX58610 15 ABX58599 15 ABX58588 15 ABX58577 15 ABX58566 15ABX58555 15 ABX58544 17 ABX58533 17 ABX58522 15 ABX58511 15 ABX58500 15ABX58489 17 ABX58478 15 ABX58467 17 ABX58456 15 ABX58445 15 ABX58434 15ABX58423 17 ABX58412 15 ABX58401 15 ABX58390 15 ABX58379 15 ABX58368 15ABX58357 17 ABX58346 15 ABX58324 15 ABX58313 17 ABX58302 15 ABX58291 15ABX58280 15 ABX58269 15 ABX58258 15 ABW91644 15 ABW91633 15 ABW91622 15ABW91611 17 ABW91600 15 ABW91589 15 ABW91578 15 ABW91567 15 ABW91545 15ABW91534 15 ABW91523 17 ABW91512 15 ABW91501 15 ABW91490 17 ABW91468 17ABW91457 17 ABW91435 15 ABW91424 15 ABW91413 17 ABW91391 15 ABW91380 15ABW91369 15 ABW91358 15 ABW91347 15 ABW91336 15 ABW91325 15 ABW91314 15ABW91303 15 ABW91281 15 ABW91226 15 ABW86614 15 ABW86549 15 ABW86538 15ABW86527 15 ABW86516 15 ABW86505 17 ABW86494 15 ABW86483 15 ABW86472 15ABW86461 15 ABW86450 15 ABW86439 15 ABW86428 15 ABW86417 15 ABW86406 17ABW86395 15 ABW86384 15 ABW86373 15 ABW86362 15 ABW86351 15 ABW86340 15ABW86329 17 ABW71478 15 ABW71467 15 ABW71456 15 ABW71445 15 ABW71434 15ABW71423 15 ABW71412 15 ABW71401 15 ABW71390 15 ABW71379 15 ABW71368 17ABW71346 15 ABW71335 15 ABW71324 15 ABW71313 17 ABW40683 15 ABW40672 17ABW40650 15 ABW40628 15 ABW40617 15 ABW40606 17 ABW40584 15 ABW40573 15ABW40562 15 ABW40551 15 ABW40540 15 ABW40529 15 ABW40518 17 ABW40507 15ABW40496 15 ABW40485 15 ABW40474 15 ABW40463 15 ABW40452 15 ABW40441 17ABW40430 15 ABW40419 15 ABW40408 15 ABW40397 15 ABW40375 15 ABW40364 15ABW40353 17 ABW40342 17 ABW40320 15 ABW40309 15 ABW40298 15 ABW40287 15ABW40265 15 ABW40243 15 ABW40232 15 ABW40221 15 ABW40210 15 ABW40188 15ABW40166 12 ABW40155 15 ABW40133 15 ABW40122 15 ABW40111 15 ABW40100 15ABW40078 15 ABW40067 15 ABW40056 15 ABW40045 15 ABW40023 17 ABW40012 15ABW40001 15 ABW39990 15 ABW39979 15 ABW39968 15 ABW39957 15 ABW39935 15ABW39924 15 ABW39913 15 ABW39902 15 ABW39891 19 ABW39869 15 ABW39858 15ABW39847 15 ABW39836 15 ABW39825 15 ABW39814 15 ABW39785 15 ABW36308 15ABW36297 15 ABW36286 15 ABW36275 15 ABW36264 15 ABW36253 15 ABW36242 15ABW36231 15 ABW36220 15 ABW36209 15 ABW36198 15 ABW36187 15 ABV82559 15ABV45967 15 ABV45956 15 ABV45945 15 ABV45934 15 ABV45923 15 ABV45901 15ABV45890 15 ABV45879 15 ABV30632 15 ABV30621 15 ABV30610 15 ABV30599 15ABV30588 15 ABV30577 15 ABV30566 15 ABV30555 15 ABV30544 15 ABV30533 15ABV30511 15 ABV30500 15 ABV30467 15 ABV30379 15 ABV30368 15 ABV30357 15ABV30346 15 ABV30335 17 ABV30324 15 ABV30313 15 ABV30302 17 ABV30291 15ABV30203 15 ABV30192 15 ABV30181 15 ABV30170 15 ABV30159 15 ABV30148 17ABV30137 15 ABV30115 12 ABV30104 15 ABV30093 15 ABV30060 15 ABV30049 15ABV30038 15 ABV30027 15 ABV30016 15 ABV30005 15 ABV29994 15 ABV29983 15ABV29972 15 ABV29961 15 ABV29950 15 ABV29928 15 ABV29895 15 ABV29884 15ABV29873 15 ABV29862 15 ABV29851 15 ABV29840 15 ABV29807 15 ABV29796 15ABV29785 15 ABV29774 15 ABV29763 15 ABV29752 15 ABV29741 15 ABV29708 15ABV29697 15 ABV29686 15 ABV29675 15 ABV29664 15 ABV29653 15 ABV29642 15ABV29620 15 ABV29609 15 ABV29587 15 ABV29576 15 ACN72614 15 P0C574 12Q20MH0 13 Q1WP00 7 ABS00328 15 Q8JSD9 10 2008 ACP20229 15 ACP20218 15ABV01075 15 ACV49684 15 66 2.0 0.2 low p < 0.001, ACU80454 15 ACU8041815 ACU80286 19 ACU80275 15 prev p < 0.05 ACU80242 18 ACU80099 15ACU80088 15 ACU80077 15 ACU80055 15 ACU80033 15 ACU79989 15 ACU12601 15ACU12590 15 ACU12579 15 ACU12568 15 ACU12513 15 ACR15466 15 ACR58560 15ACR58549 15 ACR15521 15 ACR15510 15 ACR15499 15 ACR15488 15 ACR15477 15ACR15455 15 ACR15444 15 ACR15433 15 ACR15378 15 ACR15367 15 ACR15345 15ACR15279 15 ACR15268 15 ACR15246 15 ACQ65766 15 ACP44233 15 ACP44222 15ACP44211 15 ACP44200 15 ACO95423 15 ACO95412 15 ACO95401 15 ACO95390 15ACO95379 15 ACO94878 15 ACO94867 15 ACO94713 15 ACO36405 15 ACN33153 15ACN33131 15 ACN32520 15 ACN32509 15 ACL12159 12 ACI26447 12 ACF54595 12ABV01079 15 ABV01076 15 ABV01074 15 ABV01073 15 ABV01070 15 ABV01069 15ABV01068 15 2ZNL_A 15 2009 A3DRP8 15 A4GCI3 15 A4GCK5 15 A4GBY5 8 A8C8X122 506 2.3 1.9 low p < 0.001, B3EUR4 14 A4K151 11 A4U7B4 12 A4U6W0 11A4GCL6 11 prev p < 0.002 A4GCJ4 11 A4GCM7 11 A8C8K2 15 B4URE4 12 Q0HD5211 Q289L9 15 Q07FH7 15 ACV42016 15 ACV41999 15 ACV41989 15 ACP41103 15ACV53907 15 ACV53897 15 ACV53887 15 ACV53498 15 ACV53488 15 ACV53478 14ACV53468 15 ACV53458 15 ACV53448 14 ACV53439 15 ACV41979 15 ACU30101 15ACU30091 15 ACU30081 15 ACU30071 11 ACU30017 15 ACU30007 15 ACU29997 15ACU29987 15 ACU29977 15 ACU29967 15 ACU29957 15 ACU29947 15 ACU00950 15ACU00940 15 ACU00930 15 ACT79181 15 ACT79171 15 ACT79161 15 ACT79151 15ACT79141 15 ACT22502 15 ACT21570 15 ACT11055 15 ACR54049 15 ACR47013 15ACR46669 15 ACR46660 15 ACR08608 15 ACV82595 15 ACU27039 15 ACS54299 15ACR78576 15 ACR67252 15 ACR67242 15 ACR55002 15 ACR54992 15 ACR54982 15ACR54972 15 ACR54962 15 ACR38881 15 ACR08503 15 ACS92610 15 ACU31122 15ACT36526 15 ACS73568 15 ACS73560 15 ACS73552 15 ACS69027 15 ACS36640 15ACS36639 15 ACS36637 15 ACT36534 15 ACT36503 15 ACS50086 15 ACR09394 15ACR09392 15 ACR08588 15 ACR08585 15 ACQ99679 15 ACQ99678 15 ACQ99676 15ACQ76409 15 ACQ76378 15 ACQ76357 15 ACQ76349 15 ACQ76320 15 ACQ76306 15ACQ76289 15 ACQ63280 15 ACQ63255 15 ACQ63247 15 ACQ55362 15 ACP44176 15ACP44169 15 ACP44165 15 ACP41941 15 ACR49313 15 ACR49312 15 ACR49311 15ACR49307 15 ACR49306 15 ACR20067 15 ACV71012 15 ACV71002 15 ACV70982 15ACV70972 15 ACV70962 15 ACV70952 15 ACV70942 14 ACV70932 15 ACV70922 15ACV70912 15 ACV70902 15 ACV70892 15 ACV70882 15 ACV70872 15 ACV70862 15ACV70852 15 ACV70842 15 ACV70832 15 ACV70822 15 ACV70812 15 ACV70802 15ACV70792 15 ACV70782 15 ACV70772 15 ACV70762 15 ACV70752 15 ACV70742 15ACV70732 15 ACV70722 15 ACV70712 15 ACV70702 15 ACV70692 15 ACV70682 15ACV70672 15 ACV70662 15 ACV70652 15 ACV70642 15 ACV70632 15 ACV70622 15ACV70612 15 ACV70602 15 ACV70592 15 ACV70582 15 ACV70572 15 ACV70562 15ACV70552 15 ACV70542 15 ACV70532 15 ACV70522 14 ACV70512 15 ACV70502 15ACV70492 15 ACV70482 15 ACV70472 15 ACV70462 15 ACV70452 15 ACV70442 15ACV70432 15 ACV70422 15 ACV70412 15 ACV70402 15 ACV70392 15 ACV70382 15ACV70372 15 ACV70362 15 ACV70352 15 ACV70342 15 ACV70332 15 ACV70322 15ACV70312 15 ACV70302 15 ACV70292 15 ACV70282 15 ACV70272 15 ACV70262 15ACV70252 15 ACV70242 15 ACV70232 15 ACV70222 15 ACV70212 15 ACV70202 15ACV70192 15 ACV70182 15 ACV70172 15 ACV70162 15 ACV70131 15 ACV70121 14ACV70111 15 ACV70101 15 ACV70091 15 ACV70081 15 ACV33182 15 ACV33172 15ACV33162 15 ACV33152 15 ACV33142 15 ACV33132 15 ACV33122 15 ACV33112 15ACV33102 15 ACV33092 15 ACV04587 15 ACV04577 15 ACV04567 15 ACV04557 15ACV04547 15 ACV04537 15 ACV04527 15 ACV04517 15 ACV04507 15 ACV04497 15ACV04471 15 ACV04416 15 ACV04406 15 ACV04396 15 ACV04386 15 ACV04376 15ACV04366 15 ACV04356 15 ACV04346 15 ACV04336 15 ACV04326 15 ACV04316 15ACV04306 15 ACV04296 15 ACV04286 15 ACV04276 15 ACV04266 18 ACV04256 15ACV04246 15 ACV04236 15 ACU79945 15 ACU31248 15 ACU31247 15 ACU31246 15ACU31245 15 ACU31244 15 ACU31243 15 ACU31242 15 ACU31241 15 ACU31240 15ACU31239 15 ACU31238 15 ACU31237 15 ACU27055 15 ACU27054 15 ACU17531 15ACU17461 15 ACU17399 15 ACU17332 15 ACU17269 15 ACU17198 15 ACU17136 15ACU17069 15 ACU17004 15 ACU16941 15 ACU16868 15 ACU16806 15 ACU13113 15ACU13112 15 ACU13111 15 ACU00159 15 ACT86147 15 ACT86137 15 ACT86127 15ACT86117 15 ACT86107 15 ACT86097 15 ACT86087 15 ACT86077 15 ACT86067 15ACT86057 15 ACT86047 15 ACT86037 15 ACT86027 15 ACT86017 15 ACT86007 15ACT83883 15 ACT83873 15 ACT83863 15 ACT83853 15 ACT83843 15 ACT83833 15ACT83823 15 ACT83813 15 ACT83803 15 ACR52406 15 ACT79635 14 ACT68280 15ACT68279 14 ACT68278 15 ACT68277 15 ACT68276 15 ACT68275 14 ACT68274 15ACT68273 15 ACT68272 15 ACT68271 15 ACT68270 14 ACT68269 15 ACT68268 15ACT68267 15 ACT68266 15 ACT68265 15 ACT68264 15 ACT68263 15 ACT68262 15ACT68261 15 ACR40396 15 ACT67249 15 ACT67248 15 ACT67247 15 ACT67246 15ACT67245 15 ACT67244 15 ACT67125 15 ACT67120 15 ACT21986 15 ACT21985 15ACT21984 15 ACT21983 15 ACT21982 15 ACS92608 15 ACS92587 15 ACS92577 15ACT66153 15 ACT54604 15 ACT52683 15 ACT36636 15 ACT36635 15 ACT36634 14ACT36633 15 ACT36632 15 ACS92598 15 ACT21981 15 ACT22056 15 ACT10305 15ACT09117 15 ACR67121 15 ACS78054 15 ACS78044 15 ACS78034 15 ACS78024 15ACS78014 15 ACS78004 15 ACS77994 15 ACS77984 15 ACS77974 15 ACS77964 15ACS77954 15 ACS77944 15 ACS77934 15 ACS75829 15 ACR40366 15 ACS68821 15ACS66828 15 ACS27257 15 ACS27247 15 ACS27237 15 ACS27227 15 ACS27217 15ACS27207 15 ACS27197 15 ACS14744 15 ACS14734 15 ACS14724 15 ACS14714 15ACS14704 15 ACS14694 15 ACS14684 15 ACS14674 15 ACR40386 15 ACR83536 15ACR77506 15 ACR77496 15 ACR77486 15 ACR77476 15 ACR77466 15 ACR77456 15ACR77446 15 ACR67120 15 ACR67119 15 ACR67118 15 ACR56458 15 ACR56448 15ACR56438 15 ACR56428 15 ACR56418 15 ACR56408 15 ACR56398 15 ACR56388 15ACR52496 15 ACR52486 15 ACR52476 15 ACR52466 15 ACR52456 15 ACR52446 15ACR52436 15 ACR52426 15 ACR52416 15 ACR39501 15 ACR39461 15 ACR52396 15ACR52386 15 ACR52376 15 ACR51073 15 ACR51063 15 ACR51053 15 ACR51043 15ACR51033 15 ACR51023 15 ACR51013 15 ACR51003 15 ACR40306 15 ACR40376 15ACR40356 15 ACR40346 15 ACR40336 15 ACR40326 15 ACR40316 15 ACR40296 15ACR39491 15 ACR39481 15 ACR39471 15 ACR39451 15 ACR39441 15 ACR39431 15ACR39421 15 ACR39411 15 ACR39401 15 ACR39362 15 ACR38797 15 ACR38796 15ACR38795 15 ACR18922 15 ACR15356 12 ACR10219 15 ACR10218 15 ACR10194 15ACR08589 15 ACR08587 15 ACR08584 15 ACQ84475 15 ACQ84465 15 ACR08467 15ACR08457 15 ACR08447 15 ACR08437 15 ACR08427 15 ACQ73409 15 ACQ73411 15ACQ73410 15 ACQ89953 15 ACQ89952 15 ACQ89951 15 ACQ89950 15 ACQ89949 15ACQ89948 15 ACO94845 12 ACO94834 12 ACU87262 15 ACU64816 15 ACU64797 15ACU64796 15 ACT66142 15 ACS34704 15 ACR78469 15 ACR54044 15 ACR15750 15ACR15613 15 ACV67254 15 ACV67253 15 ACV67252 15 ACV67251 15 ACV67250 15ACV67249 15 ACV67248 15 ACT35523 15 3A1G_B 1 2ZTT_B 1 A3DRP9 7 A4K152 9A4U7B5 9 A4U6W1 19 A4GCJ5 13 A4GCM8 13 A8C8K3 7 B4URE5 9 Q07FH6 7 A4GCI47 A4GBY6 7 B3EUR5 9

Example 7 Synthetic Vaccine Against H1N1

A synthetic Replikin vaccine containing an approximatelyequal-parts-by-weight mixture of eight H1N1 Replikin peptides is testedin pigs. The tested vaccine is engineered from sequences identified inH1N1 in humans from 1918 to the present and confirmed to be conserved inH1N1 over decades as well as across influenza strains with conservationparticularly noted in the key amino acid residues of the Replikinsequence, namely, lysine and histidine amino acid residues. The testedvaccine is engineered to block both the entry site of H1N1 virus and thereplication site of those H1N1 viruses that manage to enter into hostcells. As such, the vaccine is called the TWO-PUNCH vaccine. The vaccinecomprises a mixture of the following twenty Replikin peptides in sterilewater:

(SEQ ID NO: 1)  (1) HAQDILEKEHNGKLCSLKGVRPLILK; (SEQ ID NO: 2)  (2)(2) KEHNGKLCSLKGVRPLILK; (SEQ ID NO: 3)  (3)(3) KKNNAYPTIKRTYNNTNVEDLLIIWGIHH; (SEQ ID NO: 4)  (4)(4) HHSNEQGSGYAADKESTQKAIDGITNK; (SEQ ID NO: 5)  (5)(5) HDSNVKNLYDKVRLQLRDNAK; and (SEQ ID NO: 6)  (6)(6) KVRLQLRDNAKELGNGCFEFYH. (SEQ ID NO: 7)  (7) (1) KDVMESMDKEEMEITTH;(SEQ ID NO: 8)  (8) (2) HFQRKRRVRDNMTKK; (SEQ ID NO: 9)  (9)(3) KKWSHKRTIGKKKQRLNK; (SEQ ID NO: 10) (10) (4) HKRTIGKKKQRLNK;(SEQ ID NO: 11) (11) (5) HEGIQAGVDRFYRTCKLVGINMSKKK; (SEQ ID NO: 12)(12) HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK. (SEQ ID NO: 21) (13)KKGSSYPKLSKSYVNNKGKEVLVLWGVHH, (SEQ ID NO: 22) (14) HPVTIGECPKYVRSTK,(SEQ ID NO: 23) (15) KFEIFPKTSSWPNH, (SEQ ID NO: 24) (16)HNGKLCKLKGIAPLQLGK, (SEQ ID NO: 25) (17) KSYVNNKGKEVLVLWGVHH,(SEQ ID NO: 26) (18) KMNTQFTAVGKEFNH, (SEQ ID NO: 27) (19)KSQLKNNAKEIGNGCFEFYH, (SEQ ID NO: 28) (20) KIISNGTVK.

Four groups of pigs are created. The first group is a control groupwhich is neither vaccinated nor inoculated with H1N1 influenza virus.The second group is vaccinated. The third group is vaccinated andinoculated with influenza virus. The fourth group is not vaccinated butis nevertheless inoculated with H1N1 influenza virus.

The vaccine is administered to all pigs in groups 2 and 3 on days 7, 14,and 21. All pigs in groups 3 and 4 are inoculated with H1N1 on day 28.Thereafter, antibody production is monitored in the serum of selectedpigs in each group. Additionally, the pigs are monitored for symptoms ofinfluenza infections. External body fluids are also tested via PCR forshedding of H1N1 influenza. The pigs in group 2, 3, and 4 produceantibodies to H1N1. The pigs in group 2 demonstrate no symptoms ofinfluenza and shed no influenza virus detected by PCR. The pigs in group4 demonstrate significant symptoms of influenza and shed influenza virusdetected by PCR. The pigs in group 3 demonstrate reduced symptoms ofinfluenza and shed considerably less influenza virus detected by PCRthan do the pigs in group 4.

Example 8 Peptide Sequences Conserved Across Strains

Table 9 provides examples of Replikin peptides that have been identifiedas conserved in various strains of influenza.

TABLE 9 Sequences Identifiedas Conserved across  Strains Positionof first amino   Shared Shared Shared acid of in in in Replikin H5N1H9N1 H3N2 sequence pB1 pB1 pB1-F2 Conserved Replikin in Gene Gene GeneSequences H1N1 Area Area Area HYQKTMNQVVMPK   41 Yes (SEQ ID NO: 15)HCQKTMNQVVMPK   41 Yes Yes (SEQ ID NO: 14) KRWRLFSKH   78 Yes(SEQ ID NO: 16) HFQRKRRVRDNVTK  184 Yes (SEQ ID NO: 13) HFQRKRRVRDNMTK 184 Yes Yes (SEQ ID NO: 19) HFQRKRRVRDNMTKKMVTQRTIG 184 Yes KKKQRLNK (SEQ ID NO: 20) KKKHKLDK  207 Yes (SEQ ID NO: 17) KKKQRLTKX_(n=49)H²⁵³207 Yes (SEQ ID NO: 18)

Example 9 Replikin Peptide Sequences Conserved in H1N1 Isolates

The applicants surveyed hemagglutinin protein areas from H1N1 isolatesavailable at www.pubmed.com for Replikin peptides conserved between 1918and October of 2009. Applicants searched only for Replikin peptideswhere the hemagglutinin protein area of more than one isolate containedthe exact peptide (that is, a peptide that is 100% homologous withanother peptide from a different isolate). An exemplary list ofconserved Replikin peptides and the years in which isolates having theconserved Replikin peptides were identified is provided below:

-   -   1) ¹⁷⁰KKGNSYPKLSKSYINDKGKEVLVLWGIHH¹⁷⁹ (SEQ ID NO: 32) conserved        in isolates from 2009    -   2) ¹⁷⁰KNGLYPNLSKSYANNKEKEVLVLWGVHH¹⁹⁷ (SEQ ID NO: 33) observed        as conserved in isolates from 2009    -   3) KLSKSYVNNKGKEVLVLWGVHH (SEQ ID NO: 34) observed as conserved        in isolates from 1918, 1930, 1991, and 2009    -   4) KFEIFPKTSSWPNH (SEQ ID NO: 35) observed as conserved in        isolates from 1918, 1930, 1991, and 2009    -   5) KSYVNNKGKEVLVLWGVHH (SEQ ID NO: 36) observed as conserved in        isolates from 1918. 1930, 1991, 1997, 1999, 2009    -   6) HPVTIGECPKYVRSTK (SEQ ID NO: 37) observed as conserved in        isolates from 1918, 1933, 1942, 1943, 1945, 1948, 1949, 1950,        1951, 1954, 1957, 1977-1984, 1986-1989, 1991, 1994-1997,        1999-2001, 2003, 2004, 2006-2009 on the C-terminal portion of        the protein    -   7) KEFNHLEK (SEQ ID NO: 38) observed as conserved in isolates        from 1976, 1988, 1991, 1997, 1998, 2003, 2004, 2009    -   8) HLEKRIENLNKK (SEQ ID NO: 39) observed as conserved in        isolates from 1976, 1988, 1991, 1997, 1998, 2003, 2004, 2009    -   9) KMNTQFTAVGKEFNH (SEQ ID NO: 40) observed as conserved in        isolates from 1976, 1988, 1991, 1997, 1998, 2003, 2004, 2009    -   10) KHSNGTVK (SEQ ID NO: 41) observed as conserved in isolates        from 2009    -   11) KSYINDKGKEVLVLWGIHH (SEQ ID NO: 42) observed as conserved in        isolates from 2009    -   12) HPITIGKCPKYVK (SEQ ID NO: 43) observed as conserved in        isolates from 2009    -   13) KHNGKLCK (SEQ ID NO: 44) observed as conserved in isolates        from 2009    -   14) HAGAKSFYKNLIWLVKK (SEQ ID NO: 45) observed as conserved in        isolates from 2009    -   15) HKCDNTCMESVK (SEQ ID NO: 46) observed as conserved in        isolates from 2009    -   16) HSVNLLEDKHNGKLCK (SEQ ID NO: 47) observed as conserved in        isolates from 2009    -   17) HSVNILEDKHNGKLCK (SEQ ID NO: 48) observed as conserved in        isolates from 2009    -   18) KHSNGTVK (SEQ ID NO: 49) observed as conserved in isolates        from 1918, 1933, 1934, 1940, 1947, 1977, 1978, 1979, 1980, 1983,        1985    -   19) HNGKLCKLKGIAPLQLGK (SEQ ID NO: 50) observed as conserved in        isolates from 1918, 1933, 1934, 1982-1984, 2009    -   20) HNGKLCKLKGIAPLQLGK (SEQ ID NO: 51) observed as conserved in        isolates from 1918, 1933, 1934, 1982, 1983, 1984, 2009    -   21) KSQLKNNAKEIGNGCFEFYH (SEQ ID NO: 52) observed as conserved        in isolates from 2009    -   22) HPVTIGECPKYVKSTK (SEQ ID NO: 53) observed as conserved in        isolates from 1930-2008    -   23) HDSNVKNLYEKVK (SEQ ID NO: 54) observed as conserved in        isolates from 1934-2008    -   24) HDSNVKNLYEKVKSQLK (SEQ ID NO: 55) observed as conserved in        isolates from 1934-2008    -   25) HPVTIGECPKYVRSAK (SEQ ID NO: 56) observed as conserved in        isolates from 1934-2008    -   26) HKCNNECMESVK (SEQ ID NO: 57) observed as conserved in        isolates from 1940-2008    -   27) KSYVNNKEKEVLVLWGVH (SEQ ID NO: 58) observed as conserved in        isolates from 1947-2008    -   28) HPITIGECPKYVKSTK (SEQ ID NO: 59) observed as conserved in        isolates from 1976-2008    -   29) HKCDDECMESVK (SEQ ID NO: 60) observed as conserved in        isolates from 1976-2008    -   30) HNGKSSFYKNLLWLTGK (SEQ ID NO: 61) observed as conserved in        isolates from 1996-2008    -   31) HKCNDECMESVK (SEQ ID NO: 62) observed as conserved in        isolates from 1996, 2001, 2002, 2003, 2004, 2005, 2006, 2007,        2008    -   32) KSYANNKEKEVLVLWGVHH (SEQ ID NO: 63) observed as conserved in        isolates from 1999-2008    -   33) HYSRKFTPEIAK (SEQ ID NO: 64) observed as conserved in        isolates from 2000-2008    -   34) HNGESSFYRNLLWLTGKNGLYPNLSK (SEQ ID NO: 65) observed as        conserved in isolates from 2003-2008    -   35) KESWSYIVEKPNPENGTCYPGH (SEQ ID NO: 66) observed as conserved        in isolates from 2004-2008

Example 10 Conservation of SEQ ID NO: 8 in H9N2 Isolates

The applicants surveyed SEQ ID NO: 8 (HFQRKRRVRDNMTKK, originallyidentified in H5N1) in isolates of H9N2 influenza virus available atwww.pubmed.com and found the sequence in the following accession numbersat the listed positions in the following years:

Year PubMed Accession Number 1966 Q0A451 position 184, AAD49039 position184. 1976 ABB88306 position 184. 1978 AAP49097 position 184. 1979ABB20321 position 184. 1992 AAD49034 position 184. 1993 AAD49038position 184. 1994 AAD49033 position 184, AAD49032 position 184. 1995AAQ04911 position 124, AAQ04907 position 171. 1996 AAD49037 position184, AAD49036 position 184, AAQ04928 position 184, AAQ04924 position183, AAD49035 position 184. 1997 Q9WLS3 position 184, AAD49031 position184, BAB39507 position 184, BAF46425 position 184, AAD49027 position184, AAQ04927 position 184, AAQ04926 position 184, AAQ04923 position177, AAQ04920 position 180, AAQ04918 position 184, AAQ04914 position116, AAD49030 position 184, AAD49029 position 184, AAD49026 position184, CAB95863 position 184, AAD49028 position 184, AAK49362 position184, AAK49356 position 184, AAK49358 position 184, AAK49357 position184. 1998 BAB39508 position 184, AAP04506 position 184, AAQ04919position 184, AAQ04916 position 184, AAQ04910 position 171, AAL14089position 184, AAL14088 position 184, ACG59798 position 152, AAT65269position 184, AAT65267 position 184, AAT65253 position 184. 1999CAB95865 position 184, CAB95864 position 184, ABU63965 position 184,ABK59029 position 184, BAE96031 position 184, AAK49355 position 184,AAK49354 position 184, AAQ04925 position 184, AAQ04922 position 184,AAQ04915 position 179, AAQ04913 position 138, AAQ04912 position 156,AAQ04909 position 86, AAQ04908 position 124, AAL32491 position 178,ABI94772 position 184, CAC19698 position 184, AAG48199 position 170,AAG48198 position 170, AAG48196 position 170, AAG48195 position 170,AAG48194 position 170, AAG48193 position 170, AAG48192 position 170,AAG48191 position 170, AAG48190 position 170. 2000 AAP49085 position184, AAP49084 position 184, AAP49100 position 76, AAP49093 position 184,AAP49091 position 184, AAP49090 position 184, AAP49089 position 184,AAP49087 position 184, AAP49086 position 184, ABQ57376 position 184,ABF56630 position 184, ABF56639 position 184, ABF56621 position 184,ABV31865 position 184, ABV48111 position 184, ABV47990 position 184,ABV47869 position 184, ABV46313 position 184, AAQ04921 position 184,AAQ04917 position 179, AAG48197 position 170, ABB90211 position 184,ABB90200 position 184, AAN84435 position 177, AAN84404 position 177,AAN84382 position 177. 2001 AAP49099 position 179, AAP49092 position184, AAP49088 position 184, ABM46527 position 184, ABV48571 position184, ABV47605 position 173, ABV46829 position 184, ABV46626 position184, BAF46525 position 184, BAF46515 position 184, BAF46505 position184, BAF46495 position 184, BAF46465 position 184, BAF46455 position184, BAF46445 position 184, BAF46435 position 184, ABJ15707 position 87,ABI96775 position 93, ABG27054 position 184, ABG27037 position 184,ACG59796 position 152, ABM46591 position 184, ABM46590 position 184,ABM46589 position 184, ABM46588 position 184, ABM46587 position 184,ABM46586 position 184, ABM46585 position 184, ABM46584 position 184,ABM46583 position 184, ABM46582 position 184, ABM46581 position 184,ABM46580 position 184, ABM46579 position 184, ABM46578 position 184,ABM46577 position 184, ABM46576 position 184, ABM46575 position 184,ABM46574 position 184, ABM46573 position 184, ABM46572 position 184,ABM46571 position 184, ABM46570 position 184, ABM46569 position 184,ABM46568 position 184, ABM46567 position 184, ABM46566 position 184,ABM46565 position 184, ABM46564 position 184, ABM46563 position 184,ABM46562 position 184, ABM46561 position 184, ABM46560 position 184,ABM46559 position 184, ABM46558 position 184, ABM46557 position 184,ABM46556 position 184, ABM46555 position 184, ABM46554 position 184,ABM46553 position 184, ABM46552 position 184, ABM46551 position 184,ABM46550 position 184, ABM46549 position 184, ABM46548 position 184,ABM46547 position 184, ABM46546 position 184, ABM46545 position 184,ABM46544 position 184, ABM46543 position 184, ABM46542 position 184,ABM46541 position 184, ABM46540 position 184, ABM46539 position 184,ABM46538 position 184, ABM46537 position 184, ABM46536 position 184,ABM46535 position 184, ABM46534 position 184, ABM46533 position 184,ABM46532 position 184, ABM46531 position 184, ABM46530 position 184,ABM46529 position 184, ABM46528 position 184, ABM46526 position 184,ABM46525 position 184, ABM46524 position 184, ABM46523 position 184,ABM46522 position 184, ABM46521 position 184, ABM46520 position 184,ABM46519 position 184, AAN84413 position 177, AAN84412 position 177.2002 ABV47385 position 184, ABV47770 position 184, ABV47682 position184, ABV47649 position 184, ABV47550 position 184, ABV47429 position184, ABV47308 position 184, ABV47187 position 184, ABV47066 position184, BAF46485 position 184, BAF46475 position 184, ABI97312 position184, ABI94786 position 103. 2003 ABV48012 position 177, ABV48001position 184, ABV47968 position 184, ABV47957 position 184, ABV47924position 184, ABV47913 position 184, ABV47891 position 184, ABV47880position 184, ABV47858 position 184, ABV47825 position 184, ABV47814position 184, ABV47792 position 184, ABV47704 position 184, ABB58993position 184, ABB58989 position 184, ABK00142 position 184, ACA42426position 184, ABB19963 position 184, ABB58999 position 184, ABB58998position 184, ABB58997 position 184, ABB58996 position 184, ABB58995position 184, ABB58994 position 184, ABB58992 position 184, ABB58991position 184, ABB58990 position 184, AAV65823 position 184, AAU11278position 184, AAU11277 position 184, AAU11276 position 184, AAU11275position 184, AAU11274 position 184, AAU11273 position 184, AAU11272position 184, AAU11271 position 184, AAU11270 position 184, AAU11269position 184, AAU11268 position 184, AAU11267 position 184, AAU11266position 184, AAU11265 position 184, AAU11264 position 184, AAU11263position 184, AAU11262 position 184, AAU11261 position 184, AAV30834position 184, AAW78094 position 184, AAW78093 position 184, AAW78092position 184, AAW78091 position 184, AAW78090 position 184, AAW78089position 184, AAW78088 position 184, AAW78087 position 184, AAW78086position 184. 2004 ACF37318 position 184, ABL61403 position 175,ABE28411 position 184, ABV48364 position 184, ABV46895 position 184,ABV46797 position 184, ABV46787 position 184, ABV46766 position 184,ABV46670 position 184, ABV48342 position 184, ABV48331 position 172,ABV48320 position 172, ABV48287 position 171, ABV48254 position 171,ABV48221 position 184, ABV48210 position 184, ABV48144 position 177,ABV48133 position 184, ABV48122 position 184, ABV48100 position 184,ABV48089 position 184, ABV48078 position 184, ABV48067 position 184,ABV48056 position 184, ABV48045 position 184, ABV48034 position 184,ABV48023 position 172, ABV46862 position 184, ABV46776 position 184,ABV46659 position 184, ABV46648 position 184, ABV46637 position 184,ACA25366 position 184, ACA25356 position 184, ABC48846 position 184,ABC48836 position 184, ABC48826 position 184, ABC48816 position 184,ABC48806 position 148, ACA42436 position 184, AAV68029 position 184,AAV68004 position 184, AAV67998 position 184, AAV68012 position 184,AAV67990 position 184. 2005 ABS57525 position 184, ABS57521 position184, ABS57517 position 184, ACG59800 position 152, ABV48374 position184, ABV47023 position 184, ABV46958 position 184, ABV46937 position184, ABV46905 position 184, ABV31976 position 184, ABV31975 position184, ABV31955 position 184, ABV31933 position 184, ABV31913 position184, ABV31912 position 184, ABV31887 position 184, ABV48395 position184, ABV48384 position 184, ABV46915 position 184, ABQ51943 position184, ABI96712 position 184, ABV31888 position 184, ABV31851 position 1842006 ACF37316 position 184, ABV31956 position 184, ABV31935 position184, ABV31934 position 184, ABX11498 position 184. 2007 ACG59794position 152, ACG59790 position 152, ACF37320 position 184. 2008ACJ67530 position 184, ACG80390 position 184, ACG59786 position 152,ACG80392 position 184, ACJ68807 position 152.

Example 11 Conservation of SEQ ID NO: 16 in H1N1 Isolates

The applicants surveyed SEQ ID NO: 16 KRWRLFSKH in isolates of H1N1influenza virus available at www.pubmed.com and found the sequence inthe following accession numbers at the listed positions in the followingyears:

Year PubMed Accession Number 1918 id = 160417491 position 78. 1933 id =123824486 position 78. 1934 id = 119389936 position 29, id = 83288375position 78. 1935 id = 229891356 position 78, id = 133754204 position78. 1936 id = 229891357 position 78, id = 133754147 position 78. 1942 id= 89152229 position 78. 1943 id = 89903071 position 78. 1945 id =229891359 position 78, id = 145278825 position 78. 2008 id = 229891354position 78.

Example 12 Conservation of SEQ ID NO: 14 in H1N1 Isolates

The applicants surveyed SEQ ID NO: 14 (HCQKTMNQVVMPK) in isolates ofH1N1 influenza virus available at www.pubmed.com and found the sequencein the following accession numbers at the listed positions in thefollowing years:

Year PubMed Accession Number 1918 id = 160417491 position 41. 1933 id =123824486 position 41. 1934 id = 83288375 position 41. 1935 id =229891356 position 41, id = 133754204 position 41. 1936 id = 229891357position 41, id = 133754147 position 41. 1940 id = 123807038 position41, id = 112787561 position 41. 1942 id = 89152229 position 41. 1943 id= 229891358 position 41, id = 133754185 position 41, id = 133752897position 41. 1947 id = 89782408 position 41.

Example 13 Conservation of SEQ ID NO: 15 in H1N1 Isolates

The applicants surveyed SEQ ID NO: 15 (HYQKTMNQVVMPK) in isolates ofH1N1 influenza virus available at www.pubmed.com and found the sequencein the following accession numbers at the listed positions in thefollowing years:

Year PubMed Accession Number 1951 id = 229891360 position 41. 1954 id =229891361 position 41. 1977 id = 123822361 position 41. 1978 id =229891364 position 41. 1980 id = 229891365 position 41. 1983 id =229891366 position 41.

Example 14 Conservation of SEQ ID NO: 17 in H1N1 Isolates

The applicants surveyed SEQ ID NO: 17 (KKKHKLDK) in isolates of H1N1influenza virus available at www.pubmed.com and found the sequence inthe following accession numbers at the listed positions in the followingyears:

Year PubMed Accession Number 1991 id = 89112485 position 207, id =194304989 position 207. 1995 id = 218664225 position 207, id = 194304875position 207, id = 110733483 position 207, id = 109159406 position 207,id = 94959681 position 207, id = 116069900 position 207, id = 115289364position 207, id = 113170555 position 207, id = 112787646 position 207,id = 112787580 position 207, id = 110733464 position 207, id = 110733445position 207, id = 94959662 position 207, id = 109914493 position 207,id = 109159386 position 207, id = 94959624 position 207, id = 91177643position 207, id = 91124046 position 207, id = 91123250 position 207, id= 91122472 position 207, id = 91122065 position 207, id = 91121655position 207, id = 91121316 position 207. 1996 id = 89033025 position207, id = 134047404 position 207, id = 133752821 position 207, id =125664153 position 207, id = 125663990 position 207, id = 125663971position 207, id = 125663952 position 207, id = 94959700 position 207,id = 120434227 position 207, id = 120434208 position 207, id = 120434189position 207, id = 120434170 position 207, id = 116070014 position 207,id = 116069995 position 207, id = 115344695 position 207, id = 115344676position 207, id = 115344657 position 207, id = 115344638 position 207,id = 115291099 position 207, id = 112789522 position 207, id = 112789503position 207, id = 112789484 position 207, id = 112789465 position 207,id = 112789446 position 207, id = 112789427 position 207, id = 109914473position 207, id = 94959719 position 207. 1997 id = 89033028 position207. 1999 id = 89033031 position 207, id = 237689032 position 207, id =194304894 position 207, id = 117571163 position 207, id = 115607704position 207. 2000 id = 70907655 position 207, id = 157367781 position207, id = 156536333 position 207, id = 156536314 position 207, id =152963303 position 207, id = 152963284 position 207, id = 149780459position 207, id = 149780411 position 207, id = 149780392 position 207,id = 148898156 position 207, id = 148898137 position 207, id = 148898118position 207, id = 148898099 position 207, id = 145278920 position 207,id = 145278787 position 207, id = 145278633 position 207, id = 133981790position 207, id = 120433771 position 207, id = 119365477 position 207,id = 119365439 position 207, id = 118313529 position 207, id = 117571239position 207, id = 117571220 position 207, id = 117571201 position 207,id = 116070033 position 207, id = 116069919 position 207, id = 115607913position 207, id = 115607894 position 207, id = 115607761 position 207,id = 115521746 position 207, id = 115344714 position 207, id = 115344581position 207, id = 110733901 position 207, id = 110733882 position 207,id = 110629421 position 207, id = 110629402 position 207, id = 110332401position 207, id = 109914854 position 207, id = 109675833 position 207,id = 94960118 position 207, id = 94960099 position 207, id = 94960080position 207, id = 109914454 position 207, id = 109914435 position 207,id = 91119004 position 207, id = 91118985 position 207, id = 90572065position 207, id = 90572046 position 207, id = 90572027 position 207, id= 90572008 position 207, id = 90571989 position 207, id = 90571970position 207, id = 90571951 position 207, id = 90571588 position 207, id= 89787876 position 207, id = 89787681 position 207, id = 89787454position 207, id = 89787205 position 207, id = 89787093 position 207, id= 89786825 position 207, id = 89786625 position 207, id = 89786451position 207, id = 89786282 position 207, id = 89786036 position 207, id= 89785839 position 207, id = 89785636 position 207, id = 89780995position 207, id = 89780774 position 207, id = 89780574 position 207, id= 89161178 position 207, id = 89113879 position 207, id = 89113841position 207, id = 89112390 position 207, id = 89112371 position 207, id= 89112352 position 207, id = 89112333 position 207, id = 89112314position 207, id = 74477261 position 207, id = 74477242 position 207.2001 id = 149780572 position 207, id = 149780543 position 207, id =133754109 position 207, id = 133754001 position 207, id = 133752878position 207, id = 133752859 position 207, id = 133752840 position 207,id = 131058547 position 207, id = 131058280 position 207, id = 125664286position 207, id = 125664172 position 207, id = 122851250 position 207,id = 115521442 position 207, id = 110332529 position 207, id = 109675891position 207, id = 109675872 position 207, id = 109159726 position 207,id = 106896547 position 207, id = 106896528 position 207, id = 106896509position 207, id = 106896490 position 207, id = 106896471 position 207,id = 106896452 position 207, id = 106896433 position 207, id = 106896414position 207, id = 106896395 position 207, id = 106896376 position 207,id = 106896357 position 207, id = 106896338 position 207, id = 94959738position 207, id = 94959567 position 207, id = 94959548 position 207, id= 109675409 position 207, id = 106896006 position 207, id = 106895987position 207, id = 106895968 position 207, id = 94959586 position 207,id = 91125115 position 207, id = 91120873 position 207, id = 91120604position 207, id = 91120229 position 207, id = 91119868 position 207, id= 91119452 position 207, id = 91119118 position 207, id = 91119099position 207, id = 91119080 position 207, id = 91119061 position 207, id= 91119042 position 207, id = 91119023 position 207, id = 90572578position 207, id = 90572559 position 207, id = 90572540 position 207, id= 90572521 position 207, id = 90572502 position 207, id = 90572483position 207, id = 90572464 position 207, id = 90572445 position 207, id= 90572426 position 207, id = 90572407 position 207, id = 90572388position 207, id = 90572369 position 207, id = 90572350 position 207, id= 90572331 position 207, id = 90572312 position 207, id = 90572293position 207, id = 90572274 position 207, id = 90572255 position 207, id= 90572236 position 207, id = 90572217 position 207, id = 90572198position 207, id = 90572179 position 207, id = 90572160 position 207, id= 90572141 position 207, id = 90572122 position 207, id = 90572103position 207, id = 90572084 position 207, id = 90571683 position 207, id= 90571664 position 207, id = 90571645 position 207, id = 90571626position 207, id = 89789031 position 207, id = 89788757 position 207, id= 89788489 position 207, id = 89788291 position 207, id = 89112409position 207, id = 85857138 position 207, id = 83658790 position 207, id= 77746869 position 207, id = 77543658 position 207, id = 77543378position 207, id = 83314233 position 207, id = 82542615 position 207, id= 82501517 position 207, id = 82494727 position 207, id = 82494708position 207, id = 80974058 position 207, id = 77747109 position 207, id= 77747088 position 207, id = 77747069 position 207, id = 77746888position 207, id = 77543257 position 207, id = 76446835 position 205, id= 76443542 position 207, id = 76411282 position 207, id = 76366065position 207, id = 76366027 position 207, id = 75213057 position 207, id= 75171465 position 207, id = 74477301 position 207, id = 74477204position 207, id = 73765608 position 207, id = 73761573 position 207, id= 73665800 position 207, id = 71564895 position 207. 2002 id = 237689070position 207, id = 237689051 position 207, id = 237688878 position 207,id = 77543357 position 207, id = 82546790 position 207, id = 73761489position 207. 2003 id = 125664191 position 207, id = 122855965 position207, id = 89112181 position 207, id = 86806725 position 207, id =83727857 position 207, id = 77747437 position 207, id = 77543313position 207, id = 82546771 position 207, id = 82501662 position 207, id= 80974115 position 207, id = 77747475 position 207, id = 77746850position 207, id = 77746831 position 207, id = 76366046 position 207, id= 75216236 position 207, id = 75173042 position 207, id = 75171320position 207, id = 75168430 position 207, id = 74477223 position 207.2004 id = 83744850 position 207. 2005 id = 149780710 position 207, id =145278939 position 207, id = 131058820 position 207, id = 115607837position 207, id = 115607818 position 207, id = 115607799 position 207,id = 115607780 position 207, id = 115289478 position 207, id = 113170897position 207, id = 112791707 position 207, id = 112788933 position 207,id = 112788895 position 207, id = 112788876 position 207, id = 112788857position 207. 2006 id = 226954762 position 207, id = 208344099 position207, id = 161139460 position 207, id = 161138698 position 207, id =158524916 position 207, id = 157281311 position 207, id = 157281292position 207, id = 157281273 position 207, id = 118313208 position 207.2007 id = 238837391 position 207, id = 238837372 position 207, id =237688840 position 207, id = 224400125 position 207, id = 224392744position 207, id = 224027182 position 207, id = 224027163 position 207,id = 224022190 position 207, id = 224022171 position 207, id = 224022152position 207, id = 224022130 position 207, id = 224022042 position 207,id = 224020945 position 207, id = 218874700 position 207, id = 194304552position 207, id = 188504346 position 207, id = 188504008 position 207,id = 188503989 position 207, id = 183396126 position 207, id = 183396107position 207, id = 183396088 position 207, id = 169822586 position 207,id = 168480897 position 207, id = 168480878 position 207, id = 166079425position 207, id = 166079406 position 207, id = 166079387 position 207,id = 166079368 position 207, id = 166079330 position 207, id = 163964751position 207, id = 163964732 position 207, id = 163964713 position 207,id = 163964637 position 207, id = 163964618 position 207, id = 163964599position 207, id = 163964580 position 207, id = 163964561 position 207,id = 163964542 position 207, id = 163964504 position 207, id = 163964485position 207, id = 163964466 position 207, id = 163964447 position 207,id = 163964428 position 207, id = 163964409 position 207, id = 163964390position 207, id = 163964371 position 207, id = 163964333 position 207,id = 161139517 position 207, id = 161139498 position 207, id = 161139479position 207, id = 161139384 position 207, id = 161139365 position 207,id = 161139346 position 207, id = 161139327 position 207, id = 161139308position 207, id = 161139289 position 207, id = 161139270 position 207,id = 161139251 position 207, id = 161139232 position 207, id = 161139213position 207, id = 161139194 position 207, id = 161139175 position 207,id = 161139156 position 207, id = 161139137 position 207, id = 161139118position 207, id = 161139099 position 207, id = 161139080 position 207,id = 161139061 position 207, id = 161139042 position 207, id = 161139023position 207, id = 161139004 position 207, id = 161138985 position 207,id = 161138966 position 207, id = 161138947 position 207, id = 161138928position 207, id = 161138909 position 207, id = 161138890 position 207,id = 161138871 position 207, id = 161138852 position 207, id = 161138833position 207, id = 161138814 position 207, id = 161138795 position 207,id = 161138776 position 207, id = 161138757 position 207, id = 161138738position 207, id = 161138717 position 207, id = 159150270 position 207,id = 159150251 position 207, id = 159150232 position 207, id = 159150213position 207, id = 159150194 position 207, id = 159150175 position 207,id = 159150156 position 207, id = 159150137 position 207, id = 159150099position 207, id = 159150080 position 207, id = 159150061 position 207,id = 159150042 position 207, id = 159150023 position 207, id = 159150004position 207, id = 159149985 position 207, id = 159149966 position 207,id = 159149947 position 207, id = 159149909 position 207, id = 159149890position 207, id = 159149871 position 207, id = 159149833 position 207,id = 159149814 position 207, id = 159149795 position 207, id = 159149776position 207, id = 159149757 position 207, id = 159149738 position 207,id = 159149719 position 207, id = 159149700 position 207, id = 159149681position 207, id = 159149662 position 207, id = 159149643 position 207,id = 159149548 position 207, id = 158958091 position 207, id = 158957995position 207, id = 158957976 position 207, id = 158957957 position 207,id = 158957938 position 207, id = 158957919 position 207, id = 158957900position 207, id = 158957881 position 207, id = 158957862 position 207,id = 158957843 position 207, id = 158957824 position 207, id = 158957805position 207, id = 158957786 position 207, id = 158957767 position 207,id = 158957748 position 207, id = 158957729 position 207, id = 158957710position 207, id = 158957691 position 207, id = 158957672 position 207,id = 158957653 position 207, id = 158957634 position 207, id = 158957615position 207, id = 158957596 position 207, id = 158525220 position 207,id = 158525201 position 207, id = 158525182 position 207, id = 158525163position 207, id = 158525144 position 207, id = 158525125 position 207,id = 158525106 position 207, id = 158525087 position 207, id = 158525068position 207, id = 158525049 position 207, id = 158524992 position 207,id = 158524973 position 207, id = 158524954 position 207, id = 158524935position 207, id = 158454518 position 207, id = 158454499 position 207,id = 158454461 position 207, id = 158454423 position 207, id = 158454404position 207, id = 158454385 position 207, id = 158454347 position 207,id = 158454328 position 207, id = 158454309 position 207, id = 158454290position 207, id = 158454271 position 207, id = 158454252 position 207,id = 158454233 position 207, id = 158454214 position 207, id = 158454195position 207, id = 158454176 position 207, id = 158454157 position 207,id = 158454138 position 207, id = 158454119 position 207, id = 158454100position 207, id = 158454081 position 207, id = 158454062 position 207,id = 158454043 position 207, id = 158454024 position 207, id = 158453986position 207, id = 158453967 position 207, id = 158453948 position 207,id = 158453929 position 207, id = 158453891 position 207, id = 158453872position 207, id = 158453853 position 207, id = 158453834 position 207,id = 158453796 position 207, id = 158453758 position 207, id = 158453739position 207, id = 158453720 position 207, id = 158453701 position 207,id = 158453663 position 207, id = 158453625 position 207, id = 158453606position 207, id = 158453568 position 207, id = 158453549 position 207,id = 158453530 position 207, id = 158453511 position 207, id = 158453473position 207, id = 158453454 position 207, id = 158453435 position 207,id = 158453416 position 207, id = 158453378 position 207, id = 158453359position 207, id = 158453340 position 207, id = 158453321 position 207,id = 158453302 position 207, id = 158453283 position 207, id = 158453264position 207, id = 158453226 position 207, id = 158453207 position 207,id = 158453188 position 207, id = 158453169 position 207, id = 158453150position 207, id = 158453112 position 207, id = 158453093 position 207,id = 158453074 position 207, id = 158453055 position 207, id = 158453036position 207, id = 158453017 position 207, id = 158452916 position 207,id = 158344906 position 207, id = 158344887 position 207, id = 158344868position 207, id = 158344849 position 207, id = 158344830 position 207,id = 158344811 position 207, id = 158344792 position 207, id = 158344773position 207, id = 158344754 position 207, id = 158344735 position 207,id = 158344716 position 207, id = 158344697 position 207, id = 157829226position 207, id = 157368180 position 207, id = 157368161 position 207,id = 157368142 position 207, id = 157368123 position 207, id = 157368104position 207, id = 157368066 position 207, id = 157368047 position 207,id = 157368028 position 207, id = 157283155 position 207, id = 157283136position 207, id = 157283117 position 207, id = 157283098 position 207,id = 157283079 position 207, id = 157283060 position 207, id = 157283041position 207, id = 157283022 position 207, id = 157283003 position 207,id = 157282984 position 207, id = 157282946 position 207, id = 157282927position 207, id = 157282870 position 207, id = 157282718 position 207,id = 157282699 position 207, id = 157282680 position 207, id = 157282661position 207, id = 157282642 position 207, id = 157282623 position 207,id = 157282604 position 207, id = 157282585 position 207, id = 157282566position 207, id = 157282414 position 207, id = 157282395 position 207,id = 157282376 position 207, id = 157282357 position 207, id = 157282338position 207, id = 157282319 position 207, id = 157282300 position 207,id = 157282262 position 207, id = 157282243 position 207, id = 157282224position 207, id = 157282167 position 207, id = 157282148 position 207,id = 157282129 position 207, id = 157282110 position 207, id = 157282091position 207, id = 157282072 position 207, id = 157282053 position 207,id = 157282034 position 207, id = 157282015 position 207, id = 157281996position 207, id = 157281977 position 207, id = 157281939 position 207,id = 157281882 position 207, id = 157281863 position 207, id = 157281844position 207, id = 157281825 position 207, id = 157281806 position 207,id = 157281787 position 207, id = 157281730 position 207, id = 157281711position 207, id = 157281692 position 207, id = 157281673 position 207,id = 157281654 position 207, id = 157281635 position 207, id = 157281616position 207, id = 157281558 position 207, id = 157281539 position 207,id = 157281520 position 207, id = 157281501 position 207, id = 157281482position 207, id = 157281463 position 207, id = 157281444 position 207,id = 157281406 position 207, id = 157281387 position 207, id = 157281349position 207, id = 157281330 position 207, id = 224979373 position 207.2008 id = 227293800 position 207, id = 227293763 position 207, id =256386502 position 207, id = 256386426 position 207, id = 256386844position 207, id = 256386730 position 207, id = 256386483 position 207,id = 256386217 position 207, id = 256385698 position 207, id = 256385679position 207, id = 256385660 position 207, id = 256385622 position 207,id = 256385584 position 207, id = 256385527 position 207, id = 256385508position 207, id = 255529241 position 207, id = 255529222 position 207,id = 255529203 position 207, id = 255529124 position 207, id = 255529029position 207, id = 237689298 position 207, id = 238821837 position 207,id = 238821818 position 207, id = 237689393 position 207, id = 237689374position 207, id = 237689355 position 207, id = 237689336 position 207,id = 237689317 position 207, id = 237689279 position 207, id = 237689260position 207, id = 237689241 position 207, id = 237689146 position 207,id = 237689127 position 207, id = 237689089 position 207, id = 237688975position 207, id = 237688956 position 207, id = 237688916 position 207,id = 229433780 position 207, id = 227977250 position 207, id = 227977231position 207, id = 227977212 position 207, id = 227977193 position 207,id = 226957774 position 207, id = 226957755 position 207, id = 226957736position 207, id = 226957717 position 207, id = 226957698 position 207,id = 226954876 position 207, id = 226954857 position 207, id = 226954591position 207, id = 225907760 position 207, id = 224027258 position 207,id = 224027220 position 207, id = 224021250 position 207. 2009 id =256385432 position 207.

Example 15 Conservation of SEQ ID NO: 13 in H1N1 Isolates

The applicants surveyed SEQ ID NO: 13 (HFQRKRRVRDNVTK) in isolates ofH1N1 influenza virus available at www.pubmed.com and found the sequencein the following accession numbers at the listed positions in thefollowing years:

Year PubMed Accession Number-Replikin Count 1948 id = 125976177 position184. 1949 id = 125976234 position 184. 1950 id = 89114311 position 184,id = 145278806 position 184. 1951 id = 148897966 position 184, id =146760100 position 184, id = 146133749 position 184, id = 145279092position 184. 1954 id = 89112504 position 184, id = 134047499 position184. 1957 id = 86793313 position 184. 1977 id = 90572616 position 184,id = 89112466 position 184, id = 89112447 position 184, id = 133982646position 184. 1978 id = 145279034 position 184, id = 133752916 position184, id = 131059321 position 184, id = 131059087 position 184, id =125976215 position 184, id = 118313024 position 184, id = 109159444position 184, id = 94959852 position 184, id = 94959833 position 184, id= 94959814 position 184, id = 94959795 position 184, id = 94959776position 184. 1980 id = 133754166 position 184, id = 131059487 position184, id = 94959871 position 184. 1981 id = 134047461 position 184. 1982id = 90572597 position 184, id = 89782573 position 184, id = 134048464position 184. 1983 id = 159149491 position 184, id = 133754128 position184, id = 133752766 position 184, id = 131059712 position 184, id =125663865 position 184, id = 125663843 position 184, id = 122852130position 184, id = 122853012 position 184, id = 122852616 position 184,id = 120434132 position 184, id = 120434113 position 184, id = 120434094position 184, id = 120434075 position 184, id = 120434056 position 184,id = 120434037 position 184, id = 120434018 position 184, id = 120433999position 184, id = 119365572 position 184, id = 119365553 position 184,id = 118314391 position 184, id = 118314277 position 184, id = 118314219position 184, id = 117572802 position 184, id = 117572783 position 184,id = 117572764 position 184, id = 117572728 position 184, id = 117572678position 184, id = 117572220 position 184, id = 117572162 position 184,id = 117571402 position 184, id = 115289345 position 184, id = 113170574position 184, id = 112787618 position 184, id = 110733529 position 184,id = 110733510 position 184, id = 94960004 position 184, id = 94959909position 184, id = 110629007 position 184, id = 94960042 position 184,id = 94960023 position 184, id = 94959890 position 184, id = 94959947position 184, id = 109159523 position 184, id = 109159499 position 184,id = 94959985 position 184, id = 94959966 position 184, id = 94959928position 184. 1984 id = 145278863 position 184, id = 133754242 position184. 1986 id = 145278882 position 184, id = 133982626 position 184, id =133754223 position 184, id = 120434151 position 184. 1987 id = 146760005position 184, id = 125663917 position 184, id = 125663890 position 184.1989 id = 218664187 position 184. 1991 id = 89112485 position 184, id =194304989 position 184. 1995 id = 218664225 position 184, id = 194304875position 184, id = 110733483 position 184, id = 109159406 position 184,id = 94959681 position 184, id = 116069900 position 184, id = 115289364position 184, id = 113170555 position 184, id = 112787646 position 184,id = 112787580 position 184, id = 110733464 position 184, id = 110733445position 184, id = 94959662 position 184, id = 109914493 position 184,id = 109159386 position 184, id = 94959624 position 184, id = 91177643position 184, id = 91124046 position 184, id = 91123653 position 184, id= 91123250 position 184, id = 91122472 position 184, id = 91122065position 184, id = 91121655 position 184, id = 91121316 position 184.1996 id = 89033025 position 184, id = 134047404 position 184, id =133752821 position 184, id = 125664153 position 184, id = 125663990position 184, id = 125663971 position 184, id = 125663952 position 184,id = 94959700 position 184, id = 120434227 position 184, id = 120434208position 184, id = 120434189 position 184, id = 120434170 position 184,id = 116070014 position 184, id = 116069995 position 184, id = 115344695position 184, id = 115344676 position 184, id = 115344657 position 184,id = 115344638 position 184, id = 115291099 position 184, id = 112789522position 184, id = 112789503 position 184, id = 112789484 position 184,id = 112789465 position 184, id = 112789446 position 184, id = 112789427position 184, id = 109914473 position 184, id = 94959719 position 184.1997 id = 89033028 position 184. 1999 id = 89033031 position 184, id =237689032 position 184, id = 194304894 position 184, id = 117571163position 184, id = 115607704 position 184. 2000 id = 70907655 position184, id = 157367781 position 184, id = 156536333 position 184, id =156536314 position 184, id = 152963303 position 184, id = 152963284position 184, id = 149780459 position 184, id = 149780411 position 184,id = 149780392 position 184, id = 148898156 position 184, id = 148898137position 184, id = 148898118 position 184, id = 148898099 position 184,id = 145278920 position 184, id = 145278787 position 184, id = 145278633position 184, id = 133981790 position 184, id = 120433771 position 184,id = 119365477 position 184, id = 119365439 position 184, id = 118313529position 184, id = 117571239 position 184, id = 117571220 position 184,id = 117571201 position 184, id = 116070033 position 184, id = 116069919position 184, id = 115607913 position 184, id = 115607894 position 184,id = 115607761 position 184, id = 115521746 position 184, id = 115344714position 184, id = 115344581 position 184, id = 110733901 position 184,id = 110733882 position 184, id = 110629421 position 184, id = 110629402position 184, id = 110332401 position 184, id = 109914854 position 184,id = 109675833 position 184, id = 94960118 position 184, id = 94960099position 184, id = 94960080 position 184, id = 109914454 position 184,id = 109914435 position 184, id = 91119004 position 184, id = 91118985position 184, id = 90572065 position 184, id = 90572046 position 184, id= 90572027 position 184, id = 90572008 position 184, id = 90571989position 184, id = 90571970 position 184, id = 90571951 position 184, id= 90571588 position 184, id = 89787876 position 184, id = 89787681position 184, id = 89787454 position 184, id = 89787205 position 184, id= 89787093 position 184, id = 89786825 position 184, id = 89786625position 184, id = 89786451 position 184, id = 89786282 position 184, id= 89786036 position 184, id = 89785839 position 184, id = 89785636position 184, id = 89780995 position 184, id = 89780774 position 184, id= 89780574 position 184, id = 89161178 position 184, id = 89113879position 184, id = 89113841 position 184, id = 89112390 position 184, id= 89112371 position 184, id = 89112352 position 184, id = 89112333position 184, id = 89112314 position 184, id = 74477261 position 184, id= 74477242 position 184. 2001 id = 149780572 position 184, id =149780543 position 184, id = 133754109 position 184, id = 133754001position 184, id = 133752878 position 184, id = 133752859 position 184,id = 133752840 position 184, id = 131058547 position 184, id = 131058280position 184, id = 125664286 position 184, id = 125664172 position 184,id = 122851250 position 184, id = 115521442 position 184, id = 110332529position 184, id = 109675891 position 184, id = 109675872 position 184,id = 109159726 position 184, id = 106896547 position 184, id = 106896528position 184, id = 106896509 position 184, id = 106896490 position 184,id = 106896471 position 184, id = 106896452 position 184, id = 106896433position 184, id = 106896414 position 184, id = 106896395 position 184,id = 106896376 position 184, id = 106896357 position 184, id = 106896338position 184, id = 94959738 position 184, id = 94959567 position 184, id= 94959548 position 184, id = 109675409 position 184, id = 106896006position 184, id = 106895987 position 184, id = 106895968 position 184,id = 94959586 position 184, id = 91125115 position 184, id = 91120873position 184, id = 91120604 position 184, id = 91120229 position 184, id= 91119868 position 184, id = 91119452 position 184, id = 91119118position 184, id = 91119099 position 184, id = 91119080 position 184, id= 91119061 position 184, id = 91119042 position 184, id = 90572578position 184, id = 90572559 position 184, id = 90572540 position 184, id= 90572521 position 184, id = 90572502 position 184, id = 90572483position 184, id = 90572464 position 184, id = 90572445 position 184, id= 90572426 position 184, id = 90572407 position 184, id = 90572388position 184, id = 90572369 position 184, id = 90572350 position 184, id= 90572331 position 184, id = 90572312 position 184, id = 90572293position 184, id = 90572274 position 184, id = 90572255 position 184, id= 90572236 position 184, id = 90572217 position 184, id = 90572198position 184, id = 90572179 position 184, id = 90572160 position 184, id= 90572141 position 184, id = 90572122 position 184, id = 90572103position 184, id = 90572084 position 184, id = 90571683 position 184, id= 90571664 position 184, id = 90571645 position 184, id = 90571626position 184, id = 89789031 position 184, id = 89788757 position 184, id= 89788489 position 184, id = 89788291 position 184, id = 89112409position 184, id = 85857138 position 184, id = 83658790 position 184, id= 77746869 position 184, id = 77543658 position 184, id = 77543378position 184, id = 83314233 position 184, id = 82542615 position 184, id= 82501517 position 184, id = 82494727 position 184, id = 82494708position 184, id = 80974058 position 184, id = 77747109 position 184, id= 77747088 position 184, id = 77747069 position 184, id = 77746888position 184, id = 77543257 position 184, id = 76446835 position 182, id= 76443542 position 184, id = 76411282 position 184, id = 76366065position 184, id = 76366027 position 184, id = 75213057 position 184, id= 75171465 position 184, id = 74477301 position 184, id = 74477204position 184, id = 73765608 position 184, id = 73761573 position 184, id= 73665800 position 184, id = 71564895 position 184. 2002 id = 237689070position 184, id = 237689051 position 184, id = 237688878 position 184,id = 77543357 position 184, id = 82546790 position 184, id = 73761489position 184. 2003 id = 125664191 position 184, id = 122855965 position184, id = 89112181 position 184, id = 86806725 position 184, id =83727857 position 184, id = 77747437 position 184, id = 77543313position 184, id = 73763210 position 184, id = 82546771 position 184, id= 82501662 position 184, id = 80974115 position 184, id = 77747475position 184, id = 77746850 position 184, id = 77746831 position 184, id= 76366046 position 184, id = 75216236 position 184, id = 75173042position 184, id = 75171320 position 184, id = 75168430 position 184, id= 74477223 position 184. 2004 id = 83744850 position 184, id = 151335599position 184. 2005 id = 149780710 position 184, id = 145278939 position184, id = 131058820 position 184, id = 131052868 position 184, id =117572954 position 184, id = 115607837 position 184, id = 115607799position 184, id = 115607780 position 184, id = 115521499 position 184,id = 115289478 position 184, id = 113170897 position 184, id = 112791707position 184, id = 112788933 position 184, id = 112788914 position 184,id = 112788895 position 184, id = 112788876 position 184, id = 112788857position 184. 2006 id = 151335580 position 184, id = 226954762 position184, id = 208344099 position 184, id = 161139460 position 184, id =161138698 position 184, id = 158524916 position 184, id = 157281311position 184, id = 157281292 position 184, id = 157281273 position 184,id = 118313208 position 184. 2007 id = 238837391 position 184, id =238837372 position 184, id = 237688840 position 184, id = 224400125position 184, id = 224392744 position 184, id = 224027182 position 184,id = 224027163 position 184, id = 224022190 position 184, id = 224022171position 184, id = 224022152 position 184, id = 224022130 position 184,id = 224022042 position 184, id = 218874700 position 184, id = 194304552position 184, id = 188504346 position 184, id = 188504008 position 184,id = 188503989 position 184, id = 183396126 position 184, id = 183396107position 184, id = 183396088 position 184, id = 169822586 position 184,id = 168480897 position 184, id = 168480878 position 184, id = 166079425position 184, id = 166079406 position 184, id = 166079387 position 184,id = 166079368 position 184, id = 166079330 position 184, id = 163964751position 184, id = 163964713 position 184, id = 163964637 position 184,id = 163964618 position 184, id = 163964599 position 184, id = 163964580position 184, id = 163964561 position 184, id = 163964542 position 184,id = 163964504 position 184, id = 163964485 position 184, id = 163964466position 184, id = 163964447 position 184, id = 163964428 position 184,id = 163964409 position 184, id = 163964390 position 184, id = 163964371position 184, id = 163964333 position 184, id = 161139517 position 184,id = 161139498 position 184, id = 161139479 position 184, id = 161139384position 184, id = 161139365 position 184, id = 161139346 position 184,id = 161139327 position 184, id = 161139308 position 184, id = 161139289position 184, id = 161139270 position 184, id = 161139251 position 184,id = 161139232 position 184, id = 161139213 position 184, id = 161139194position 184, id = 161139175 position 184, id = 161139156 position 184,id = 161139137 position 184, id = 161139118 position 184, id = 161139099position 184, id = 161139080 position 184, id = 161139061 position 184,id = 161139042 position 184, id = 161139023 position 184, id = 161139004position 184, id = 161138985 position 184, id = 161138966 position 184,id = 161138947 position 184, id = 161138928 position 184, id = 161138909position 184, id = 161138890 position 184, id = 161138871 position 184,id = 161138852 position 184, id = 161138833 position 184, id = 161138814position 184, id = 161138795 position 184, id = 161138776 position 184,id = 161138757 position 184, id = 161138738 position 184, id = 161138717position 184, id = 159150270 position 184, id = 159150251 position 184,id = 159150232 position 184, id = 159150213 position 184, id = 159150194position 184, id = 159150175 position 184, id = 159150156 position 184,id = 159150137 position 184, id = 159150099 position 184, id = 159150080position 184, id = 159150061 position 184, id = 159150042 position 184,id = 159150023 position 184, id = 159150004 position 184, id = 159149985position 184, id = 159149966 position 184, id = 159149947 position 184,id = 159149909 position 184, id = 159149890 position 184, id = 159149871position 184, id = 159149833 position 184, id = 159149814 position 184,id = 159149795 position 184, id = 159149776 position 184, id = 159149757position 184, id = 159149738 position 184, id = 159149719 position 184,id = 159149700 position 184, id = 159149681 position 184, id = 159149662position 184, id = 159149643 position 184, id = 159149548 position 184,id = 158958091 position 184, id = 158957995 position 184, id = 158957976position 184, id = 158957957 position 184, id = 158957938 position 184,id = 158957919 position 184, id = 158957900 position 184, id = 158957881position 184, id = 158957862 position 184, id = 158957843 position 184,id = 158957824 position 184, id = 158957805 position 184, id = 158957786position 184, id = 158957767 position 184, id = 158957748 position 184,id = 158957729 position 184, id = 158957710 position 184, id = 158957691position 184, id = 158957672 position 184, id = 158957653 position 184,id = 158957634 position 184, id = 158957615 position 184, id = 158957596position 184, id = 158525220 position 184, id = 158525201 position 184,id = 158525182 position 184, id = 158525163 position 184, id = 158525144position 184, id = 158525125 position 184, id = 158525106 position 184,id = 158525087 position 184, id = 158525068 position 184, id = 158525049position 184, id = 158525030 position 184, id = 158524992 position 184,id = 158524973 position 184, id = 158524954 position 184, id = 158524935position 184, id = 158454518 position 184, id = 158454499 position 184,id = 158454461 position 184, id = 158454423 position 184, id = 158454404position 184, id = 158454385 position 184, id = 158454347 position 184,id = 158454328 position 184, id = 158454309 position 184, id = 158454290position 184, id = 158454271 position 184, id = 158454252 position 184,id = 158454233 position 184, id = 158454214 position 184, id = 158454195position 184, id = 158454176 position 184, id = 158454157 position 184,id = 158454138 position 184, id = 158454119 position 184, id = 158454100position 184, id = 158454081 position 184, id = 158454062 position 184,id = 158454043 position 184, id = 158454024 position 184, id = 158453986position 184, id = 158453967 position 184, id = 158453948 position 184,id = 158453929 position 184, id = 158453891 position 184, id = 158453872position 184, id = 158453853 position 184, id = 158453834 position 184,id = 158453796 position 184, id = 158453758 position 184, id = 158453739position 184, id = 158453720 position 184, id = 158453701 position 184,id = 158453663 position 184, id = 158453606 position 184, id = 158453568position 184, id = 158453549 position 184, id = 158453530 position 184,id = 158453511 position 184, id = 158453473 position 184, id = 158453454position 184, id = 158453435 position 184, id = 158453416 position 184,id = 158453378 position 184, id = 158453359 position 184, id = 158453340position 184, id = 158453321 position 184, id = 158453302 position 184,id = 158453283 position 184, id = 158453264 position 184, id = 158453226position 184, id = 158453207 position 184, id = 158453188 position 184,id = 158453169 position 184, id = 158453150 position 184, id = 158453112position 184, id = 158453093 position 184, id = 158453074 position 184,id = 158453055 position 184, id = 158453036 position 184, id = 158453017position 184, id = 158452916 position 184, id = 158344906 position 184,id = 158344887 position 184, id = 158344868 position 184, id = 158344849position 184, id = 158344830 position 184, id = 158344811 position 184,id = 158344792 position 184, id = 158344773 position 184, id = 158344754position 184, id = 158344735 position 184, id = 158344716 position 184,id = 158344697 position 184, id = 157829226 position 184, id = 157368180position 184, id = 157368161 position 184, id = 157368142 position 184,id = 157368123 position 184, id = 157368104 position 184, id = 157368066position 184, id = 157368047 position 184, id = 157368028 position 184,id = 157283155 position 184, id = 157283136 position 184, id = 157283117position 184, id = 157283098 position 184, id = 157283079 position 184,id = 157283060 position 184, id = 157283041 position 184, id = 157283022position 184, id = 157283003 position 184, id = 157282984 position 184,id = 157282946 position 184, id = 157282927 position 184, id = 157282870position 184, id = 157282718 position 184, id = 157282699 position 184,id = 157282680 position 184, id = 157282661 position 184, id = 157282642position 184, id = 157282623 position 184, id = 157282604 position 184,id = 157282585 position 184, id = 157282566 position 184, id = 157282414position 184, id = 157282395 position 184, id = 157282376 position 184,id = 157282357 position 184, id = 157282338 position 184, id = 157282319position 184, id = 157282300 position 184, id = 157282243 position 184,id = 157282224 position 184, id = 157282167 position 184, id = 157282148position 184, id = 157282129 position 184, id = 157282110 position 184,id = 157282091 position 184, id = 157282072 position 184, id = 157282053position 184, id = 157282034 position 184, id = 157282015 position 184,id = 157281977 position 184, id = 157281939 position 184, id = 157281882position 184, id = 157281863 position 184, id = 157281844 position 184,id = 157281825 position 184, id = 157281806 position 184, id = 157281787position 184, id = 157281730 position 184, id = 157281711 position 184,id = 157281692 position 184, id = 157281673 position 184, id = 157281654position 184, id = 157281635 position 184, id = 157281616 position 184,id = 157281558 position 184, id = 157281539 position 184, id = 157281520position 184, id = 157281501 position 184, id = 157281482 position 184,id = 157281463 position 184, id = 157281444 position 184, id = 157281406position 184, id = 157281387 position 184, id = 157281349 position 184,id = 157281330 position 184, id = 224979373 position 184. 2008 id =227293800 position 184, id = 227293763 position 184, id = 256386502position 184, id = 256386426 position 184, id = 256386844 position 184,id = 256386730 position 184, id = 256386483 position 184, id = 256386217position 184, id = 256385698 position 184, id = 256385679 position 184,id = 256385660 position 184, id = 256385622 position 184, id = 256385584position 184, id = 256385527 position 184, id = 256385508 position 184,id = 255529241 position 184, id = 255529222 position 184, id = 255529203position 184, id = 255529124 position 184, id = 255529029 position 184,id = 237689298 position 184, id = 238821837 position 184, id = 238821818position 184, id = 237689393 position 184, id = 237689374 position 184,id = 237689355 position 184, id = 237689336 position 184, id = 237689317position 184, id = 237689279 position 184, id = 237689260 position 184,id = 237689241 position 184, id = 237689146 position 184, id = 237689127position 184, id = 237689089 position 184, id = 237688975 position 184,id = 237688956 position 184, id = 237688916 position 184, id = 229433780position 184, id = 227977250 position 184, id = 227977231 position 184,id = 227977212 position 184, id = 227977193 position 184, id = 226957774position 184, id = 226957755 position 184, id = 226957736 position 184,id = 226957717 position 184, id = 226957698 position 184, id = 226954876position 184, id = 226954857 position 184, id = 226954591 position 184,id = 225907760 position 184, id = 224027258 position 184, id = 224027220position 184, id = 224021250 position 184. 2009 id = 256385432 position184.

What is claimed is:
 1. An isolated or chemically synthesized peptideconsisting of 7 to 50 amino acid residues and comprising at least onepeptide A that is at least 90% 3-0% homologous with at least one of SEQID NO(s): 1-20. 2-4. (canceled)
 5. The isolated or chemicallysynthesized peptide of claim 1 wherein said at least one peptide Aconsists of at least one of SEQ ID NO(s): 1-20.
 6. The isolated orchemically synthesized peptide of claim 1 consisting of at least one ofSEQ ID NO(s): 1-20.
 7. The isolated or chemically synthesized peptide ofclaim 1 consisting of at least one of SEQ ID NO(s): 1-12. 8-9.(canceled)
 10. The isolated or chemically synthesized peptide of claim 1consisting essentially of at least one of SEQ ID NO(s): 1-20.
 11. Theisolated or chemically synthesized peptide of claim 1 consistingessentially of at least one of SEQ ID NO(s): 1-12.
 12. The isolated orchemically synthesized peptide of claim 1 wherein said peptide A is atleast 95% homologous with at least one of SEQ ID NO(s): 1-20. 13-18.(canceled)
 19. An immunogenic or blocking composition comprising atleast one peptide of claim
 1. 20. The immunogenic or blockingcomposition of claim 19 comprising at least one peptide of claim 6.21-24. (canceled)
 25. The immunogenic or blocking composition of claim19 comprising at least two isolated or chemically synthesized peptidesof SEQ ID NO(s): 1-20.
 26. The immunogenic or blocking composition ofclaim 19 comprising a mixture of peptides, wherein the mixture comprisesisolated or chemically synthesized peptides of each of SEQ ID NO(s):1-12.
 27. (canceled)
 28. The immunogenic or blocking composition ofclaim 26, wherein the mixture comprises approximately equal weight ofthe isolated peptides of each of SEQ ID NO(s): 1-12.
 29. The immunogenicor blocking composition of claim 28 comprising about 10% by weight SEQID NO: 1, about 9% by weight SEQ ID NO: 2, about 10% by weight SEQ IDNO: 3, about 6% by weight SEQ ID NO: 4, about 8% by weight SEQ ID NO: 5,about 8% by weight SEQ ID NO: 6, about 7% by weight SEQ ID NO: 7, about6% by weight SEQ ID NO: 8, about 10% by weight SEQ ID NO: 9, about 8% byweight SEQ ID NO: 10, about 7% by weight SEQ ID NO: 11, and about 11% byweight SEQ ID NO: 12, wherein the percent by weight is based on theweight of said mixture of peptides.
 30. The immunogenic or blockingcomposition of claim 19 further comprising a pharmaceutically acceptableexcipient. 31-39. (canceled)
 40. An antibody or antibody fragment thatbinds to at least a portion of an amino acid sequence of at least onepeptide of claim
 1. 41. The antibody or antibody fragment of claim 40that binds to at least a portion of a peptide consisting of at least oneof SEQ ID NO(s): 1-20. 42-53. (canceled)
 54. A method of preventing ortreating influenza virus infection comprising administering at least oneisolated or chemically synthesized peptide of claim 1 to an animal orhuman.
 55. (canceled)
 56. The method of claim 54 comprisingadministering at least one isolated or chemically synthesized peptide ofSEQ ID NO(s): 1-20 to an animal or human. 57-95. (canceled)
 96. Theisolated or chemically synthesized peptide of claim 1, wherein saidpeptide is chemically synthesized by solid phase methods.
 97. Theisolated or chemically synthesized peptide of claim 1, wherein saidpeptide is dissolved in water.